Background : The kinetic and metabolic disposition of diazepam have been studied by measuring its concentration in plasma or serum. However, the informations available on the urinary excretions of diazepam and its metabolites in human are limited. Hence, to facilitate the investigation of the metabolic disposition of diazepam employing human urine samples, a simple high-performance liquid chromatographic method was developed to method diazepam and its major metabolites. N-desmethyldiazepam, temazepam and oxazepam in urine, and used this method in a preliminary study of diazepam metabolism in 6 healthy male volunteers. Methods : One milliliter of urine, acetate buffer and ${eta}$-glucuronidase/sulfatase were incubated. After, dichlormethane was added, the sample was extracted by shaking and centrifugation. Then, re-extraction was conducted with adding of hexane, HCI and dichlormethane. The residue of the extract was dissolved in the mobile phase and then was injected into the HPLC apparatus. Results : The assasy allows simultaneous determination of all analytes in 1 mL of urine and can be used to detect 2 ng/mL with a signal-to-noise ratio of 3. Recoveries of the analytes and the internal standard (prazepam) were > 82.9%. Intra- and interassay coefficients of variation for all analytes were <4.8 and 8.7% at 10 ng/mL, and they were <2.4 and 5.5% at 100 ng/mL, respectively. Relative intra- and interassay errors ranged from -7.7 to +7.3 at 10 ng/mL. and from -1.9 to +0.6% at 100 ng/mL. The mean(${pm}SD$) cumulative urinary excretions of diazepam, desmethylidiazepam, temazepam and oxazepam over 96 h after on oral administration of 4 mg of diazepam were $0.27{pm}0.12,;3.89{pm}0.36,;6.64{pm}1.41;and;2.76{pm}0.56%$ of the dose, respectively. Conclusion: Thus, the present method is more practical and precise to investigate the diazepam metabolism non-invasively in a large number of volunteers and patients.