Among diverse protein post-translational modifications (PTMs), 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, the 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 OGT substrates discovery. Melting curves of proteins under different treatments were profiled and compared with high reproducibility and consistency. Conse-quently, proteins with significant shifted stabilities caused by OGT and UDP-GlcNAc were screened out, from which new O-GlcNAcylated proteins were uncovered.