The quest for new and enhanced vaccines requires improved means for identification of relevant antigens and the epitopes present within these. While CD8+ T cells recognize antigenic peptides presented in the context of MHC Class I, and play a key role in cellular immunity, CD4+ helper T-cells recognize antigens in the context of MHC Class II and assist other immune cells in orchestration of the defined immune response. Being a functional assay, ELISPOT can be used to define both MHC Class I and II epitopes as well as to validate them. Providing the quality of the cells used in the assay is sufficiently good, ELISPOT provides a solid platform for both screening and validating T-cell epitopes. While some ELISPOT assays rely upon loading of dendritic cells with antigen followed by incubation of HLA matched or autologous PBMC or enriched T cells, the assay presented here uses PBMC from diseased or vaccinated individuals to simultaneously identify and validate T-cell epitopes.