Protein O-GlcNAcylation, an extensive post-translational modification (PTM), has been proposed as a nutrient and stress sensor that regulates multiple essential cellular processes. Due to the low stoichiometry of O-GlcNAcylation and the high complexity of biological samples, comprehensive analysis of O-GlcNAcylation is an extraordinary challenge. In this work, we established a novel, simple, site-specific O-GlcNAc identification strategy by integrating bioorthogonal ligation and tryptic-cleavage，and applied it for O-GlcNAc proteomics analysis and applications. In this strategy, the capture step was based on click chemistry reaction between solid material and peptides, and the release step was completed by efficient tryptic cleavage. Benefiting from the simplified workflow and a tryptic-cleavable tag, this strategy would be a versatile tool for the proteomic analysis of O-GlcNAcylation to explore O-GlcNAcylation functions involving in cellular physiology and pathology disease.