Core fucosylation and O-GlcNAcylation are two most famous protein glycosylations and regulate diverse physiological and pathological processes in living organism. Core fucosylation is a protein glycosylation in which a L-fucose residue attaches to the innermost N-acetylglucosamine (GlcNAc) of N-glycan through α1, 6 linkage. O-GlcNAcylation is a dy-namic protein glycosylation where D-GlcNAc moieties attaches to serine or threonine residues of proteins. Here, a “two-birds one stone” strategy is described for site-specific analysis of the core fucosylation and O-GlcNAcylation. The de-scribed strategy allows the simultaneously profiling of core-fucosylated glycoproteome and O-GlcNAcylated glycoprote-ome from one complex sample.