Objective: In this study, a simple and reliable high performance liquid chromatographic method with UV detection was developed and validated for rapid determination of coumarin hydroxylase activity in rat hepatic microsomes. Materials and Methods: The chromatographic separation was achieved using Zorbax Eclipse XDB C18 column (150×4.6 mm, 5 μm), which was kept at 40°C. The isocratic mobile phase consisted of methanol and 1% glacial acetic acid mixture (35:65, v/v) with a flow rate of 0.6 ml/min. The effluent was monitored at 320 nm using photodiode array detector (PDA). 6-hydroxychlorzoxazone (6-OH CZX) was used as internal standard. Results and Conclusion: The method exhibited good linearity (R2 >0.999) for both coumarin (COUM) and its 7-hydroxy metabolite (7-OH COUM) over the assayed concentration range (0.025-5.0 μM) and demonstrated good intra-day and inter-day precision and accuracy (relative standard deviations and the deviation from predicted values were less than 15%). The detection limits were 0.001 and 0.005 μM for coumarin and 7-hydroxycoumarin, respectively. This method was also successfully applied for studying the effect of three phytochemicals on hepatic CYP2A6 activity in rats.