A new, simple, rapid, sensitive, and repeatable isocratic reverse-phase HPLC method was developed and validated for simultaneous determination of midazolam and its main three hydroxylated metabolites, i.e. 1'-hydroxymidazolam, 4-hydroxymidazolam, and 1',4-dihydroxymidazolam in rat liver perfusate and also plasma. Diazepam was used as an internal standard to ensure precision and accuracy of this method. Analytes were extracted from alkalinized samples into diethyl ether using single-step liquid-liquid extraction. A C18 analytical column and a mobile phase composed of acetonitrile and sodium acetate buffer were used for the chromatographic separation with UV detection. Limits of detection varied between 7.9 and 19.6 microg/L for midazolam and its hydroxy metabolites. The overall recovery for the analytes exceeded 92%, for concentrations twice the limits of detection. The intra- and inter-day precision at three different concentrations never exceeded 8 and 11% variation, respectively. This method is applicable for modeling and description of possible pharmacological interactions on rat (CYP3A1/2) or human (CYP3A4/5) cytochrome P450 enzymes.