A high performance liquid chromatographic (HPLC) assay was developed for the simultaneous quantitation of midazolam (MDZ) and ketoconazole (KTZ) in plasma. MDZ, KTZ and diazepam (internal standard) were extracted from 100 microL or 500 microL plasma from rat or human, respectively, using liquid-liquid extraction with diethyl ether in the presence of 0.1N NaOH. After vortexing, centrifugation and freezing, the organic layer was transferred to clean tubes and evaporated. The dried residue was reconstituted in mobile phase and injected into the HPLC through a C18 column. The mobile phase consisted of acetonitrile:15 mM potassium dihydrogen orthophosphate (45:55, v/v), pumped at 1 mL/min and measured at lambda=220 nm. The method was tested in a pharmacokinetic study involving orally dosed KTZ 40 mg/kg in 1% methylcellulose followed by intravenous dosing of 5mg/kg MDZ to rats 1.5h latter. The components eluted within 10 min and were baseline resolved with no interferences from endogenous substances in plasma. The calibration curves were linear (r(2)=0.999) over the range of 25-25,000 and 5-10,000 ng/mL of KTZ and MDZ in rat and human plasma, respectively. The intraday and interday CV% were <15% and <6% for KTZ and <7% and <4% for MDZ and the mean error was <13% for both drugs in rat plasma. In human plasma the intraday CV% and % error of the mean were <11% and <10% for KTZ, respectively; both values were <13% for MDZ. The validated lower limit of quantitation was 25 and 5 ng/mL for both drugs based on 100 muL rat plasma and 500 microL human plasma, respectively. In rats, plasma concentrations of MDZ and KTZ were simultaneously measured up to 8 and 9.5h, respectively. In conclusion, the assay was shown to be rapid, sensitive and appropriate for use in drug-drug interaction studies involving MDZ and KTZ in rat, and potentially in humans.