Killing of cancer cells by cytotoxic Tcells requires efficient presentation of tumor antigens by human leukocyte antigen (HLA) molecules and patient-specific HLA genotypes also influence response to immune checkpoint inhibitors. Genotyping for a specific HLA allele can be used as biomarker to predict the immunotherapy outcome of a patient. Traditionally, HLA genotyping is achieved by sequencing the genome or transcriptome extracted from whole cells. Unfortunately, this approach does not reflect the actual abundance of each expressed HLA allele that presents antigen on the cell surface or provide any information on the post-translational modification of HLA proteins. To tackle this problem, we present an integrated immunopeptidomics and proteomics approach to comprehensively characterize the HLA proteins. HLA proteins are captured from traditional immunopeptidomics analysis and subjected to digestion, glycopeptides enrichment, and mass spectrometric analysis. Database with the mass spectrometric data will provide the relative expression level of each HLA allele and its pattern of glycosylation, which improves the understanding of antigen presentation.