Mucin-type O-glycans on glycoproteins in animal cells play important roles in many biological processes. Core 1 β3galactosyltransferase (Core 1 β3GalT, T-synthase) is a key enzyme in the O-glycan biosynthetic pathway. Emerging evidence has shown the importance of O-glycans and the absolute requirement of T-synthase in this pathway. The assessment of the T-synthase activity has historically been conducted using a radioactive method. Here we describe a fluorescence-based assay procedure for T-synthase activity. T-synthase utilizes the acceptor substrate 4-methylumbelliferone-α-GalNAc (GalNAcα-(4-MU)) and the donor substrate UDP-Gal to synthesize the disaccharide product Galβ1,3GalNAcα-(4-MU) structure. This product is specifically hydrolyzed by endo-α-N-acetylgalactosaminidase (O-glycosidase) releasing free 4-MU. Free 4-MU is highly fluorescent at pH 9.6-10 and can be easily measured by a fluorescent detector (Ex: 355 nm; Em: 460 nm). This fluorescence-based T-synthase assay is simple, sensitive, reproducible, not affected by enzyme source, and adaptable for high-throughput assays.