Among diverse protein post-translational modifications, O-GlcNAcylation, a simple but essential monosaccharide modification, plays crucial roles in cellular processes and is closely related to various diseases. Despite its ubiquity in cells, properties of low stoichiometry and reversibility are hard nuts to crack in system-wide research of O-GlcNAc. Herein, we developed a novel method employing multi-comparative thermal proteome profiling for O-GlcNAc transferase (OGT) substrate discovery. Melting curves of proteins under different treatments were profiled and compared with high reproducibility and consistency. Consequently, proteins with significantly shifted stabilities caused by OGT and uridine-5'-diphosphate N-acetylglucosamine were screened out from which new O-GlcNAcylated proteins were uncovered.