This study focused on HLA-A24 and comprehensively analyzed the ligandome of colon and lung cancer cells without the use of MHC-binding in silico prediction algorithms. Affinity purification using the antibody specific to HLA-A24 followed by LC–MS/MS sequencing was used to detect peptides, which harbored the known characteristics of HLA-A24 peptides in terms of length and anchor motifs. Ligandome analysis demonstrated the natural presentation of two different types of novel tumor-associated antigens. The ligandome contained a peptide derived from SUV39H2, a gene found to be expressed in a variety of cancers but not in normal tissues (except for the testis). The SUV39H2 peptide is immunogenic and elicits cytotoxic CD8⁺ T-cell (CTL) responses against cancer cells and is thus a novel cancer-testis antigen. Moreover, we found that microsatellite instability (MSI)-colon cancer cells displayed a neoepitope with an amino-acid substitution, while microsatellite stable (MSS)-colon and lung cancer cells displayed its counterpart peptide without the substitution. Structure modeling of peptide-HLA-A24 complexes predicted that the mutated residue at P8 was accessible to T-cell receptors. The neoepitope readily elicited CTL responses, which discriminated it from its wild-type counterpart, and the CTLs exhibited considerably high cytotoxicity against MSS-colon cancer cells carrying the responsible gene mutation. The specific and strong CTL lysis observed in this study fosters our understanding of immune surveillance against neoantigens.