A high-performance liquid chromatography method is described which allows for the simultaneous determination of antipyrine, 4-hydroxyantipyrine, norantipyrine and 3-hydroxyantipyrine. Hydrolysis conditions with respect to source of glucuronidase, amount of glucuronidase, pH of incubation and addition of antioxidant to the incubation medium proved to be very critical for norantipyrine and to a lesser extent 4-hydroxyantipyrine. The method is of good sensitivity, specificity, accuracy and reproducibility. Thus, the method is well suited for detailed metabolic studies.