Malignant transformation is often accompanied by an aberrant glycosylation profile of the cell surface—in particular, the production of GlcNAcβ1-6Manα1 branches in N-linked glycoproteins. To identify the target glycoproteins, we show a method using recombinant chicken N-acetylglucosaminyltransferase VI (GnT VI) and radiolabeled uridine (5′-)diphosphate-GlcNAc. The assay exploits the fact that GnT VI has a strict requirement for the GlcNAcβ1-6Manα1 structure for activity, when a pyridylaminated free N-glycan is used as the acceptor substrate. Human asialo-agalacto α1-acid glycoprotein (AGP), which is known to contain GlcNAcβ1-6Manα1 branches in its N-linked glycan chains, was radiolabeled when reacted with GnT VI, whereas human asialo-agalacto transferrin and bovine fetuin, neither of which contains a GlcNAcβ1-6Manα1 structure were not, thus corroborating the specificity of the assay. Several proteins from human serum after pretreatment with sialidase and β-galactosidase could be detected using the assay. One was identified as AGP from its mobility on SDS–PAGE, demonstrating the potential of this assay even with crude materials. Furthermore, this method could detect a protein that was also positively stained with leukoagglutinating phytohemagglutinin (L4-PHA) using glycoproteins prepared from WiDr human colon cancer cells. This method should provide a useful complement to the current method, which relies on the specificity of L4-PHA.