Abstract: In an in vitro method of determining the modification degree of O-GlcNAc in a biological sample, a first sample is treated with B3GALNT2 to incorporate a GalNAz into closed O-GlcNAc sites in the sample, labeled using click chemistry to form labeled closed O-GlcNAc sites, and the labeled closed O-GlcNAc sites in the first sample are detected (corresponding to the number of closed O-GlcNAc sites in the biological sample). A second, duplicate sample is treated with OGT to O-GlcNAcylate open O-GlcNAc sites in the sample to convert the open O-GlcNAc sites to closed O-GlcNAc sites, and treated with B3GALNT2 to incorporate GalNAz into the closed O-GlcNAc sites. The second sample is labeled using click chemistry to form labeled closed O-GlcNAc sites (corresponding to the total number of O-GlcNAc sites in the biological sample). The number of closed O-GlcNAc sites and total O-GlcNAc sites in the biological sample are compared to determine the modification degree of O-GlcNAc.