O-linked β-N-acetylglucosamine (O-GlcNAc) modification is a dynamic post-translational modification that involves the reversible attachment of N-acetylglucosamine to serine or threonine residues of nuclear, cytoplasmic, and mitochondrial proteins. This modification is critical for regulating cellular processes such as signal transduction, transcription, translation, protein degradation, and cellular localization. Aberrant O-GlcNAcylation has been implicated in numerous pathological conditions, including diabetes, cardiovascular diseases, neurodegenerative disorders, and cancers, highlighting the need for precise methods to study its dynamics across biological systems.