Immune surveillance of infected or tumor cells by CD8+ T cells requires that MHC class I molecules present a diverse repertoire of peptides on the cell surface. Even a few copies of individual peptides among this mixture are sufficient for recognition by the antigen receptors of appropriate CD8 T cells. Here we describe methods for biochemical analysis of the naturally processed peptides and their precursors in living cells. The peptides are fractionated using reverse phase high performance liquid chromatography and detected by the activation of CD8+ T cell hybridomas. The results provide information on the structure and amount of the peptides and yield insights into the mechanisms that generate the naturally processed peptides.