O-linked-β-N-acetylglucosamine (O-GlcNAc) is a dynamic PTM of the 3′-hydroxyl groups of serine or threonine residues of nuclear, cytoplasmic, and mitochondrial proteins. The cycling of this modification is regulated in response to nutrients, stress, and other extracellular stimuli by the catalytic activities of O-GlcNAc transferase and O-GlcNAcase. O-GlcNAc is functionally similar to phosphorylation and has been demonstrated to play critical roles in numerous biological processes, including cell signaling, transcription, and disease etiology. Since its discovery nearly 30 years ago, studies have demonstrated that the O-GlcNAc is highly abundant and widespread, like phosphorylation however, the development of methodologies to study O-GlcNAc at the site level has been challenging. Recently, a number of studies have overcome these challenges and describe new tagging, enrichment, and mass spectrometric-based approaches to study O-GlcNAc in terms of its site identification, stoichiometry, and dynamics on proteins. The development of these methods are key for elucidation of O-GlcNAc's functional crosstalk with phosphorylation and other PTMs, and will serve to provide the necessary information for the development of site-specific antibodies, which will aid in the determination of a particular protein's site-specific function. In this review, we describe these methods and summarize results obtained from them demonstrating the roles of O-GlcNAc in diabetes, cancer, Alzheimer's, and in learning and memory, while also describing how these new strategies have implicated O-GlcNAc as a potential diagnostic for the screening of patients for prediabetes.