Specific immunotherapy of cancer utilizes tumor‐directed cytotoxic T lymphocytes (CTL) that lyse tumor cells presenting MHC class I‐associated peptides derived from tumor‐associated proteins. Many tumor‐associated gene products are known, but corresponding T cell epitopes are only known for relatively few of these. The most commonly used approaches to identify such antigens require pre‐existing CTL lines or clones. By using a CTL‐independent high performance liquid chromatography mass spectrometry (HPLC MS)–based approach we identified HLA‐A2‐presented peptides from carcinoembryonic antigen and wild‐type p53 with a copy number as low as eight molecules per cell. Potential epitopes were predicted from the sequences of known tumor antigens and the corresponding synthetic peptides were analyzed by nanocapillary HPLC MS. In parallel, peptides were extracted from fresh, solid tumor tissue or tumor cell lines and analyzed in the same way. Upon co‐elution of a natural peptide with a predicted peptide of the same mass, the peptide sequence was confirmed by on‐line tandem MS. This approach allows rapid screening of large numbers of tumor‐associated gene products for naturally processed peptides presented by different MHC class I molecules as a prerequisite for efficient epitope identification and rapid transfer to therapeutic vaccine trials.