The presentation of neoantigens on the cell membrane is the foundation for most cancer immunotherapies. Due to their extremely low abundance, analyzing neoantigens in clinical samples is technically difficult, hindering the development of neoantigen-based therapeutics for more general use in the treatment of diverse cancers worldwide. Here, we describe an integrated system, “Valid-NEO”, which reveals patient-specific cancer neoantigen therapeutic targets from minute amounts of clinical samples through direct observation, without computer-based prediction, in a sensitive, rapid, and reproducible manner. The overall four-hour procedure involves mass spectrometry analysis of neoantigens purified from tumor samples through recovery of HLA molecules with HLA antibodies. Valid-NEO could be applicable to the identification and quantification of presented neoantigens in cancer patients, particularly when only limited amounts of sample are available.