A new unified assay for the determination of UDP-glucuronosyltransferase (UGT) activities has been developed. The resolution of [14C]uridine diphosphate glucuronic acid from radiolabeled glucuronides formed by incorporation of this radiolabel can now be achieved by a sensitive and rapid-gradient HPLC method which utilizes a radioactivity endpoint as a universal detection method. One important application of this method is the determination of kinetic parameters for cloned and expressed UGT isoforms with greater speed and precision than can be afforded by TLC methodology. Moreover, assays with 14C-labeled substrates indicate that gradient HPLC can easily resolve the substrate from the glucuronide products and present an alternative to the time-consuming optimization of conditions for organic phase extraction assays.