UDP-N-acetylglucosaminyl transferase (OGT) catalyzes O-linked glycosylation of cytosolic and nuclear proteins, but enzyme studies have been hampered by the lack of a rapid, sensitive, and economical OGT assay. Employed assay methods typically involved the use of HPLC, formic acid, and large amounts of expensive radiolabeled [3H]UDP-N-acetylglucosaminyl ([3H]UDP-GlcNAc). In the current study, we have developed an OGT assay that circumvents many of these problems through four critical assay improvements: (1) identification of an abundant and enriched source of OGT enzyme (rat brain tissue), (2) utilization of a rapid method for efficiently removing salts and sugar nucleotides from cytosol (polyethylene glycol precipitation of active enzyme), (3) expression of a recombinant p62 acceptor substrate designed to facilitate purification (polyhistidine metal-chelation site), and (4) development of two alternative methods to rapidly separate free [3H]UDP-GlcNAc from 3H-p62ST acceptor peptide (trichloroacetic acid precipitation and metal-chelation affinity purification). To study the enzymology of OGT, independent of potential regulatory proteins within cytosol, we also developed and characterized an alternate OGT assay that uses antibody-purified OGT as the enzyme source. The major advantage of this assay lies in the ability to measure OGT in the absence of other cytosolic proteins.