Mutations in DNA mismatch repair genes such as MLH1 and MSH2 lead to DNA replication errors that cannot be repaired, resulting in microsatellite instability (MSI). Around 15% of colorectal cancers (CRC) exhibit high-frequency microsatellite instability (MSI-H) with a consequent lack of effective therapies. Tumour-specific antigens, or neoantigens, are ideal targets for T cell mediated immunotherapy. The MSI-H phenotype is associated with high mutational burden and subsequently increased potential sources of neoantigen-derived peptide antigens. We aimed to identify neoantigens isolated from human leucocyte antigens (HLA) of patient-derived MSI-H CRC cells, HCA7 and HCT116, using mass spectrometry. Cell surface HLA class ǀ peptide complexes were isolated from interferon-gamma (IFN- γ) treated and untreated CRC cell lines. The peptide repertoire was interrogated by LC-MS/MS using a Bruker timsTOF PRO 2 mass spectrometer coupled to a nanoElute UHPLC. Using Peaks Online 11, data was searched against human proteome appended with a cell line specific mutation database derived from exome sequencing. We identified 87571 HLA-A*02:01-bound peptides that included 65 neopeptides in untreated cells and a further 8 additional neopeptides found only upon IFN-γ treatment. Among these 73 neopeptides, 4 were shared between the two CRC cell lines. In addition, another 79523 HLA class ǀ peptides were identified across the remaining HLA allotypes expressed by these cells, including 27 neopeptides in untreated cells and 8 found in IFN-γ treated neopeptides. Of note, a high proportion of frameshift mutations were observed compared to missense mutations within the neopeptide sequences. The peptides identified, methodology applied and IFN-γ effect have implications for future development of peptide-based vaccines and adoptive T cell therapies in CRC.