Immunopeptidome is the repertoire of peptides bound to human leukocyte antigen (HLA), the allelic types of which vary from races and populations. With the improvement in mass spectroscopic techniques and bioinformatic analysis, increasing numbers of immunopeptidome database have been established worldwide. Few studies have yet been published focusing on the alleles with high frequency among Chinese, which consists of 20% human population. Last year, our group initiated the Chinese Immunopeptidome Project in the virtue of high throughput sample preparation and mass spectrometry for immunopeptidome profiling. In this project, small amount of peripheral blood was collected from 250 volunteers to generate immortalized B cell lines for HLA-binding peptide identification, as well as genomic and transcriptomic analyses. The database established in the project was also utilized to facilitate machine learning based models for neoantigen prediction and discovery. Meanwhile, we have developed a protocol to validate naturally processed and presented epitopes from clinical specimens with targeted mass spectrometric (parallel reaction monitoring) approach, which serves as the basis to following investigation on affinity and immunogenicity of HLA-binding peptide in vitro. Altogether, the database and the protocol will contribute to the current anti-cancer therapeutic interventions and shed light on the implementation of multi-omics study in cancer immunology and system biology.