Protein O-GlcNAcylation, involving the attachment of single N-acetylglucosamine (GlcNAc) to the hydroxyl group of serine or threonine residues. Elucidation of O-GlcNAcylation sites on proteins is required in order to decipher its crucial roles in regulating cellular processes and aid in drug design. With an increasing number of O-GlcNAcylation sites identified by mass spectrometry (MS)-based proteomics, several methods have been proposed for the computational identification of O-GlcNAcylation sites. However, no development that focuses on the investigation of O-GlcNAcylated substrate motifs has existed. Thus, we were motivated to design a new method for the identification of protein O-GlcNAcylation sites with the consideration of substrate site specificity.