A method for the synthesis and purification of large quantities of four radiolabeled substrates for quantitation of uncovering enzyme is described. Four substrates, [3H]GlcNAc-alpha-P-Man alpha Me, [3H]GlcNAc-alpha-P-uteroferrin, [3H]GlcNAc alpha-P-Man alpha 1-2Man-O-Me, and [3H]GlcNAc alpha-P-Man9GlcNAc, were enzymatically synthesized using GlcNAc-phosphotransferase from Acanthamoeba castellanii and uridine diphosphate N-acetyl-[3H]glucosamine and, as acceptor, methyl-alpha-D-mannopyranoside (Man alpha Me), uteroferrin, Man alpha 1-2Man-O-methyl, or Man9GlcNAc. The isolation of the [3H]GlcNAc-P-modified product of each reaction is detailed. Two assays for the detection of uncovering enzyme activity using [3H]GlcNAc-alpha-P-uteroferrin and [3H]GlcNAc-alpha-P-Man alpha Me are outlined. The ability to easily synthesize four relevant substrates for uncovering enzyme offers flexibility in assaying uncovering enzyme.