Introduced as a minor antagonist and a sidekick to Quinn Fabray (Dianna Agron) in the first episode of Glee, Santana's role grew over the course of the show's first season.

In the second season, Rivera was promoted to a series regular, and Santana was given more high-profile storylines, such as the development of her romantic feelings for her best friend Brittany Pierce (Heather Morris), and the subsequent realization that she is a lesbian.

Santana is introduced alongside Quinn and Brittany as one of the three most popular cheerleaders at the fictional William McKinley High School in Lima, Ohio, where the show is set.

In season two, the show delves into the motivations behind Santana's antagonism and her sexual aggressiveness towards boys; it is later revealed that her cruel attitude is partly driven by her struggle with her romantic feelings towards Brittany, and subsequently with her lesbian identity.

[3] Initially romantically linked to football player Puck, Santana soon breaks up with him over his bad credit score,[4] though the two have an on-again, off-again relationship well into the show's second season.

When the two are accused of giving Sue the New Directions set list for their first show choir competition, Santana defends herself and admits that she has come to enjoy participating in the club.

[7] When Puck briefly dates Mercedes Jones (Amber Riley), Santana is jealous and confronts her with an aggressive duet of "The Boy Is Mine".

[10] While making out, Brittany suggests to Santana that the two of them should sing Melissa Etheridge's "Come To My Window" for a glee club duet competition.

[13] Confronted by Brittany about her feelings for her, Santana confesses that she is in love with her but fears being ostracized by the student body for being in a same-sex relationship, due to what had happened to Kurt (Chris Colfer), who is openly gay, so many times that year.

[16] Santana is briefly banished from New Directions for disloyalty, and though she is soon allowed to return, she leaves again to join Mercedes in a newly formed all-girl glee club, the Troubletones, and convinces Brittany, who is now her girlfriend, to come with her.

When her supposed apology to Finn becomes another stream of insults, he accuses her of being a coward for tearing other people down because she can't admit that she's in love with Brittany, thus outing her in a crowded public hallway.

In the season finale, after the New Directions win Nationals in Chicago, Santana's mother (Gloria Estefan) writes her a check so she can pursue her dreams in New York if she really wants to.

After a jealous unsuccessful attempt to break up her exes Sam and Brittany in "Diva", Santana realizes she belongs in New York with Rachel and Kurt and moves in with them.

When Santana found out that Rachel is stuck in Los Angeles because of her audition for a television show, she suggests that she would play Fanny Brice for the night.

Along with Quinn and Brittany, she performs "Problem" by Ariana Grande in an attempt to recruit cheerleaders to join the revamped New Directions, currently being revived by Rachel.

Before the ceremony, Sue arrives with Alma, who she has helped to realize that although she may not believe women should marry each other, family is the most important thing, leading her and Santana to reconcile.

[23] Rivera drew on her own high school experience of unpopularity to prepare for the role, as well as watching films such as Mean Girls to "really get in the zone and feel like a bitchy Sophomore".

When a promotional clip for the episode "Sectionals" indicated that Brittany and Santana had slept together, Dorothy Snarker, writing for lesbian entertainment website AfterEllen.com, praised the pairing, referring to them by the portmanteau "Brittana".

Snarker called the two her "new favorite Glee pairing", commenting that: "While Heather Morris (Brittany) and Naya Rivera (Santana) have had minimal screen time, they’ve made it count.

Sandra Gonzalez of Entertainment Weekly was somewhat surprised by the storyline, observing that Santana had not previously been portrayed as "so serious and vulnerable", but enjoyed the depth it brought to her character.

The reveal about Santana hasn't washed away her less savory qualities; indeed, it's heightened them, to a degree, as she struggles to be true to herself and still maintain her status as the hottest girl in school.

[44] Rolling Stone contributor Erica Futterman wrote that the performance was one of Glee's best sequences: "Mercedes and Santana nail their vocals and the song combines great tracks from one of the year's biggest albums while capturing both the sass and sadness of the Troubletones at this particular moment.

"[45] Billboard editor Rae Votta called it the best performance since the cast's rendition of "Don't Stop Believin'" in the season one finale, "Journey to Regionals", and stated, "Naya Rivera's powerful vocals and the strong arrangement still packs the emotional punch Glee was targeting.

[58] The impact of the character spread through pop culture, with Jorge Rodriguez-Jimenez of Mitú writing that "You didn't have to be an avid viewer of the show to understand and appreciate the magnitude of Santana Lopez".

[67] Romero noted that while Santana starts the show as little more than "the feisty Latina trope", Rivera's performance gave her more nuance and the character ultimately confronted others for perpetuating harmful Latino stereotypes in the episode "The Spanish Teacher"; this episode notably included a duet between Rivera and the queer Latino Ricky Martin, something Romero describes as one of the show's more subtle nods to queer representation and "one of Santana's best moments of Latinx pride".

[56] Ramírez also highlighted Santana's confidence in performing in musical theatre as positive representation, showing her "unapologetically taking up room in [a] predominantly white space".

Digital Spy's Emily Browne wrote of the significance of showing Santana's emotional response to her sexuality but not being defined by it, with the character being "a popular, confident, smart femme".

[69] A BBC retrospective in 2020 looked at the impact of Rivera's role as Santana in providing important representation, internationally, for young queer women and the idea of being out in the music industry.

[70] Following Rivera's death in July 2020, numerous outlets and individuals cited her portrayal of Santana as a positive representation of both Latinas and the LGBT community in the media.

Naya worked hard to give that gift to so many",[71] and then-California senator Kamala Harris wrote in a tweet that Rivera inspired "countless young LGBTQ+ people to unapologetically be themselves".