A real-time kinetic method for measuring the activity of microsomal p-nitrophenol hydroxylase, in which the rate is measured directly by uv-visible spectrophotometry, is described. The method is based on the fact that the reaction product, 4-nitrocatechol, absorbs at 480 nm and longer wavelengths while the absorbance of the reactant, p-nitrophenol, decreases to baseline at these wavelengths. The conditions of the assay are similar to the incubation conditions of the Reinke and Moyer method. The advantages of the new method include simplicity, direct measurement of the rate rather than use of a timed assay, and elimination of experimental steps such as changing pH and centrifugation before spectrophotometric reading. The new method produces results that are comparable to and may be more reproducible than those of the Reinke and Moyer method.