A fast and reliable method for the assay of UDP-glucuronosyltransferase (UGT) activity toward aglycones containing a carboxylic acid function is described. The procedure involves incubation with UDP-[U-14C]-glucuronic acid, the common substrate for the reaction, solid-phase separation of the radiolabeled acylglucuronides and unreacted cofactor, and quantification by liquid scintillation counting. The technique was validated for each of the seven substrates tested by reversed-phase HPLC, and was then applied successfully to the determination of optimal conditions for the activation of the carboxylic acid-UGT, and the estimation of kinetic constants for the glucuronidation of clofibric acid, 2-naphthylacetic acid, naproxen, and 4,4,4-triphenylbutanoic acid in rat liver microsomes. From the results obtained, we believe that this is an assay which, with only minor modification, could be applied to a wide range of carboxylic acid substrates for which, until now, specific and sensitive assays have been largely unavailable.