O-GlcNAcylation is a dynamic protein post-translational modification of serine or threonine residues by an O-linked monosaccharide N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation was discovered three decades ago and its significance has been implicated in several disease states, such as metabolic diseases, cancer and neurological diseases. Yet it remains technically challenging to characterize comprehensively and quantitatively because of its low abundance, low stoichiometry and extremely labile nature under conventional collision-induced dissociation tandem MS conditions. Herein, we review the recent advances addressing these challenges in developing proteomic approaches for site-specific O-GlcNAcylation analysis, including specific enrichment of O-GlcNAc peptides/proteins, unambiguous site-determination of O-GlcNAc modification and quantitative analysis of O-GlcNAcylation.