On 1 April 1623, at the age of nine, he entered the choir of the Church of San Luigi dei Francesi in Rome, where he completed his studies with Vincenzo Ugolini.
In 1647 at the invitation of Cardinal Jules Mazarin, Pasqualini stayed in Paris, where he participated in the creation of the opera Orfeo by his composer Luigi Rossi on a libretto by Francesco Buti.
From 1631/2 Pasqualini was a protagonist of many operas produced at the Palazzo Barberini and Teatro delle Quattro Fontane.
He benefited greatly from the generosity of his patrons, the Barberini family of Pope Urban VIII, who were enthusiastic supporters of early opera.
Contemporary testimony leaves little doubt that the "veritable passion" the cardinal felt extended to more than Pasqualini's beautiful voice.