The O-N-acetyl-β-D-glucosaminylation, termed O-GlcNAcylation, is an atypical glycosylation corresponding to the transfer of a unique saccharide, the N-acetyl-β-D-glucosamine, on the hydroxyl group of serine and threonine amino acids of nuclear, cytosolic and mitochondrial proteins. Because of its dynamism, its reversibility and the swiftness of GlcNAc moieties transfer, O-GlcNAcylation is akin to phosphorylation and it is able to respond rapidly to both extracellular and intracellular demands. This post-translational modification is highly represented on intracellular proteins (in particular cytosolic, nuclear and mitochondrial proteins), and is involved in almost if not all cellular processes. O-GlcNAcylation is nowadays clearly associated with the etiology of several acquired diseases, in particular diabetes, neuro-degenerative disorders, cardiovascular diseases or cancer. The O-GlcNAcylation rapidly emerged as a major cellular mechanism which compete with phosphorylation in terms of modified proteins and the importance in cellular physiology. The O-GlcNAcylated sites could also correspond to phosphorylated ones; many proteins are modified by both O-GlcNAc and phosphates groups, and these two post-translational modifications could compete to the same or at neighboring sites. However, despite the undeniable role of O-GlcNAcylation in the modulation of almost all cellular processes, its precise function remains still misunderstood, mainly because of the unknown position of O-GlcNAcylation on specific domains of proteins. Thus, the precise localization of O-GlcNAcylated sites remains an indispensable prerequisite for the fine understanding of its biological function. However, mapping the O-GlcNAcylated sites remains laborious, but challenging, because of (i) the low stoichiometry of O-GlcNAcylation; (ii) the ion suppression of the modified peptide by the unmodified peptide present in larger excess; and (iii) the labile β bound between serine or threonine and the O-GlcNAc moiety which is broken during the CID (Collision-Induced Dissociation) fragmentation process, leading to loss of site information. To respond to this biological demand and to overcome these difficulties, enrichment of O-GlcNAc modified proteins and the use of other fragmentation processes like ECD (Electron Capture Dissociation), ETD (Electron Transfer Dissociation), or HCD (High-energy Collisional Dissociation), able to limit the O-GlcNAc loss during the fragmentation, are crucial. Our strategy was an alternative, specific, efficient and selective purification of O-GlcNAc bearing proteins from skeletal muscle cells by the use of the Click chemistry methodology. To extensively map O-GlcNAc sites on proteins, we proposed herein an intensive fractionation of the muscle cell proteome according to solubility, hydrophobicity and isoelectric point of proteins. The method of Click chemistry was achieved on each enriched-fraction containing proteins of interest.