The knowledge of tumor-associated T-cell epitopes is important for the understanding of tumor biology and the development of cancer vaccines. We describe here a biochemical approach for the identification of tumor-associated T-cell epitopes. Peptides are extracted from immunoaffinity isolated MHC class I molecules of tumor cells and separated by HPLC. The HPLC fractions are then tested for biological activity of the peptides which are then sequenced by mass spectrometry. The tumor association of the identified T-cell epitopes is confirmed using synthetic analogs and T-cells of cancer patients.