Mammalian UDP-glucuronosyltransferases are a family of isoenzymes that catalyze the reaction of endobiotics and xenobiotics with glucuronic acid resulting in the formation of hydrophilic glucuronides. This pathway is an important step in the metabolism and subsequent excretion of many compounds that would otherwise have toxic effects. This unit describes three methods for measuring UGT activity. Thin layer chromatography is a powerful screening method and may be used to analyze multiple substrates simultaneously. The Sep-Pak C18 cartridge extraction method has been developed to specifically separate opioid glucuronides from UDP-glucuronic acid. Finally, the ethyl acetate extraction method is used to separate the glucuronides of bilirubin, sterols, and vile acids from UDP-glucuronic acid. These methods may be applied to a microsomal fraction or to cultured cells transformed with cDNA for UGT.