A rapid and specific high-performance liquid chromatographic assay was developed for the determination of acetaminophen glucuronide formed by human liver microsomes. In addition, incubation conditions were systematically evaluated. Conditions that yielded the optimal rate of acetaminophen glucuronide formation over various concentrations of acetaminophen (0.15-30 mM) consisted of the following: 0.1 M potassium phosphate buffer, 1 mM magnesium chloride, 30 microg/mg alamethicin, 4 mM uridine 5'-diphosphoglucuronic acid at a pH of 7.1. Alamethicin produced higher and more consistent APAPG formation rates compared to Brij-58. Adding saccharolactone to the incubation medium reduced the velocity of the reaction. Acetaminophen glucuronide, acetaminophen, and the internal standard (paraxanthine), were analyzed on a C18 column with UV detection at 250 nm. The mean correlation coefficient (r2) of the standard curves for acetaminophen glucuronide was >0.99 over the range of 0.1-25 nmol. The intra- and inter-day coefficients of variation were <4%. This method is suitable for in vitro studies using acetaminophen glucuronide formation as an index reaction for UGT activity.