The development of new vaccines against pathogens is an important part of infectious disease control. In the last decade, a variety of proteins giving rise to naturally processed pathogen-derived antigenic peptides, representing B-cell and T-cell epitopes, have been characterized. Numerous candidate vaccines consisting of synthetic peptides are being designed and evaluated, with encouraging results. In this context, the application of mass spectrometry based on the isolation and identification of pathogen-derived peptides from the human leukocyte antigen (HLA) molecules is a major focus of peptide-based vaccine development. Dramatic improvements have been made in mass spectrometer performance for peptide sequencing in terms of increased sensitivity, the ability to rapidly obtain data-directed tandem mass spectra, and the accuracy of mass measurement. This review focuses on the efforts to identify T-cell epitopes for viral and microbial pathogens for directed vaccine development.