Here, we report an O-glycopeptide truncation strategy for the characterization of protein O-GalNAcylation in biological samples. The O-glycopeptide truncation strategy utilizes another protease or O-glycopeptidase for targeted cleavage of tryptic and enriched O-glycopeptides, which increases the analytical coverage of O-GalNAc glycopeptides and glycoproteins. Tryptic O-glycopeptides covered with dense O-glycan clusters and terminal sialic acids could be well isolated by the hydrophilic-based enrichment approaches. And the enriched O-glycopeptides are then enzymatically truncated into shorter or less multiply O-glycosylated peptides, which are more favorable for MS detection and database search in general bottom-up glycoproteomics. We exploit different enzymatic schemes in the O-glycopeptide truncation strategy for large-scale O-glycoproteome analysis. Ultimately, we comprehensively identify nearly 2000 O-glycopeptides from 391 glycoproteins in a total of 75 µL human serum. Moreover, the truncated O-glycopeptides contribute to site-specific O-glycosylation characterization in stepped high-energy collisional dissociation fragmented LC-MS/MS. Together, the O-glycopeptide truncation strategy has great potential to facilitate the in-depth study of O-GalNAc glycoproteomics in biological samples.