Glycosylation and phosphorylation are two of the most important forms of post-translational modification. Their dysregulation is related to tumorigenesis with phosphorylating enzymes, known as kinases, having been shown to active cancerous signaling pathways, and aberrant glycosylation has been detected in various types of cancers. For immunotherapy using neoantigens, both glycosylated and phosphorylated peptides presented exclusively on tumor cells by the major histocompatibility complex (MHC) are promising candidates due to their specificity to elicit cytotoxic T-cell responses. Unlike other MHC-presented peptides, glycosylated and phosphorylated MHC peptides cannot be predicted by bioinformatic tools, and their identification relies on specific enrichment from the whole immunopeptidome and sensitive detection with mass spectrometry. Herein, we describe a workflow that sequentially enriches glycosylated and phosphorylated MHC peptides from the immunopeptidome with hydrophilic interaction chromatography and titanium dioxide nanoparticles, followed by analysis of enriched peptides by liquid chromatography tandem mass spectrometry and identification of modified MHC peptides by database searching.