We report here a new pH-indicator-based assay for galactosyltransferase. The method is simple and fast, requires no specialized equipment, labeled substrate, or other expensive materials, and is thus expected to have broad applications including automated high-throughput screening. The method is based upon the detection of absorbance change of a pH indicator, phenol red, in response to proton release that accompanies the galactosyltransferase-catalyzed galactose transfer. The assay was used to compare three galactosyltransferases in our collection. As demonstrated here, subtle differences in substrate specificity were readily discerned with this sensitive method. All three enzymes accept both N-acetylglucosamine and glucose as acceptor but the relative activity varies with the origin of the enzyme. The method was demonstrated to be useful in the initial characterization of recombinant galactosyltransferase from crude cell extract. Optimal metal cofactor Mn(2+) concentration and temperature were determined with the method. Overall, the method offers a great improvement over current methods in reducing time and material consumption. It is the first pH-sensitive method for galactosyltransferase. The principles of using pH indicator in galactosyltransferase assay should be applicable to other glycosyltransferase enzymes.