O-linked N-acetylglucosamine (O-GlcNAc) is a monosaccharide posttranslational modification that modifies serine/threonine residues of nucleocytoplasmic proteins in metazoans. O-GlcNAc, like phosphorylation, is dynamic and responsive to numerous stimuli in diverse regulatory pathways. O-GlcNAc may also be found adjacent to or at the same sites as phosphorylation, demonstrating the potential for a reciprocal function on some of these proteins. Like most posttranslational modifications, O-GlcNAc is substoichiometric and may be found at multiple sites with other posttranslational modifications present. Additionally, there is no consensus sequence defining the addition of O-GlcNAc to the peptide backbone, further complicating identification and site mapping. This chapter describes several strategies to confirm that proteins are O-GlcNAc modified and provide subsequent determination of O-GlcNAc attachment sites. We have listed the strengths and limitations of each protocol to allow readers to decide which suits their system and availability of resources. These protocols include galactosyltransferase labeling, immunoblotting, using mass spectrometry based on beta-elimination followed by Michael addition with dithiothreitol, and chemoenzymatic labeling, enrichment, and detection.