[citation needed] Longini began his career with the International Center for Medical Research and Training and the Universidad del Valle in Cali, Colombia, where he worked on tropical infectious disease problems and taught courses in biomathematics.

He has worked on the analysis of epidemics of influenza, HIV, tuberculosis, cholera, dengue fever, malaria, rhinovirus, rotavirus, measles and other infectious agents.

[3] Longini develops mathematical statistical methods to estimate the transmission and natural history of infectious diseases.

Longini extensively studied the transmission and of pandemic and interpandemic influenza and its control with antiviral agents and vaccines.

Longini investigated how infectious diseases such as influenza,[5] cholera, typhoid and dengue could be controlled with vaccines.