This paper describes a continuous spectrophotometric assay for glycosyltransferases. In this assay, a nucleotide diphosphate is coupled to NADH oxidation via pyruvate kinase and lactate dehydrogenase. The nucleotide diphosphate is produced either directly during the glycosyltransferase mediated reaction, or indirectly by the production of a nucleotide monophosphate during the glycosyltransferase mediated reaction, and subsequent conversion of the nucleotide monophosphate to nucleotide diphosphate using nucleoside monophosphate kinase. Using this assay, kinetic parameters for fucosyl-, sialyl-, and N-acetylglucosaminyltransferases were determined. The assay not only allows continual monitoring of the enzymatic reaction, but is rapid and allows the processing of 96 samples at once since it is performed in 96-well microtiter plates. In addition, the procedure provides a means of monitoring the activity of these enzymes using sugar-nucleotide donor analogs, where radiochemical procedures cannot be used.