Platinum (Pt)-based chemotherapeutics is emerging as a promising strategy for cancer treatment in clinical trials. Protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification has been reported to occur on several important proteins implicated in regulating cancer resistance to Pt drugs. However, the O-GlcNAc proteomic landscape during this process remains poorly characterized. Herein, we report quantitative profiling of O-GlcNAcylation sites with carboplatin exposure, by using a chemoenzymatic labeling-assisted chemoproteomic approach. The stoichiometric differences of 169 O-GlcNAcylated sites are quantified, many of which occur on essential genome stability regulators. Furthermore, we discover that the cellular O-GlcNAc level is elevated upon carboplatin treatment and suppression of O-GlcNAcylation sensitizes cancer cells to carboplatin-induced cytotoxicity. These results establish a valuable resource for elucidating the function role of O-GlcNAcylation in drug resistance and shed new light on cancer chemotherapy. The chemoproteomic strategy should be generally applicable for monitoring O-GlcNAc dynamic changes in various pharmacological processes.