The accurate identification of tumor neoantigens, particularly cryptic antigens from non-canonical translation, is crucial for developing effective cancer immunotherapies. However, the significance and action of cryptic antigens in anti-tumour immunity remain unclear. In this study, we utilized a highly sensitive mass spectrometry-based de novo sequencing approach to identify non-canonical MHC-binding immunopeptides and evaluate the anti-tumor immune effects of vaccines composed of these peptides in Hepatocellular carcinoma (HCC).