Circulating CD8+ T cells (CTL) survey the peptide/MHC class I complexes on cell surface to discriminate self and eliminate foreign/transformed cells. Thus, the MHC class I peptide repertoire directly influences cells fate, and provides information for immunotherapy strategy. Here we target HLA-A24 (A24), the most frequent serotype in Asia, and perform large-scale mass spectrometric profiling of natural peptides. Using the antibody specific to A24, we identified 264 peptides from colon (SW480, Colo320, HCT15/b2m) and 331 peptides from lung (LHK2, Sq-1) cancer cells. Although, known A24 binding anchors (Y/F at P2 and F/L/I at P9) are strongly conserved among the detected ligands, a subset of peptides with an unusual anchor (K/R at the C-terminal P9 or P10) is observed, suggesting diverse usage of anchors in certain types of cancer cells. Moreover, some peptides (and their genes) are exclusively expressed in cancer stem cells (cells able to exclude Hoechst dye). In summary, we identified approx. 500 non-overlapping natural peptides presented by A24 from colon and lung cancer cells. A combination of natural peptides specific to tumors could be an ideal way for a CTL-based immunotherapy.