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 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, accuracy and reproducibility. Thus, the method is well suited for detailed metabolic studies.