The essential human O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme responsible for modifying thousands of intracellular proteins with the monosaccharide O-GlcNAc. This unique modification plays crucial roles in human health and disease, but the substrate recognition of OGT remains poorly understood. Intriguingly, the only human enzyme reported to remove this modification, O-GlcNAcase (OGA), is O-GlcNAc modified. Here, we exploited a GlcNAc electrophilic probe (GEP1A) to rapidly screen OGT mutants in a fluorescence assay that can discriminate between altered OGT-sugar and -protein substrate binding to help elucidate the binding mode of OGT toward OGA protein substrate. Since OGT tetratricopeptide repeat (TPR) domain plays a key role in OGT-OGA binding, we screened 30 OGT TPR mutants, which revealed 15 "ladder like" asparagine or aspartate residues spanning TPRs 3-7 and 10-13.5 that affect OGA O-GlcNAcylation. By applying a truncated OGA construct, we found that OGA's N-terminal region or pseudo histone acetyltransferase domain is not required for its O-GlcNAcylation, suggesting OGT functionally interacts with OGA through its catalytic and/or stalk domains. This work represents the first effort to systemically investigate each OGT TPR and our findings will facilitate the development of new strategies to investigate the role of substrate-specific O-GlcNAcylation.