A method determining the plasma concentration of clotiazepam was developed by using gas chromatography/mass spectrometry with an ion-trap detector and was validated for applying pharmacokinetics to human volunteers orally taken 5 mg dose of clotiazepam. The detection limit was 1 ng/ml and the limit of quantitation was 5 ng/mt. Intraday reproducibility and accuracy bias % were less than 8.2 and 10.2% with inter-day variations for those being within 7.0 and 13.8%, respectively. The recovery of clotiazepam was higher than 87%. The principal pharmacokinetic parameters were determined from the plasma concentration-time plot by non-compartmental or two-compartmental analysis. In non-compartmental analysis, the elimination half-life of 10.4 hr and the area under the curve of 651.3 ng hr/ml were determined, and the maximal concentration (158.6 ng/ml) in the plasma was obtained at 0.56 hr post-dose. The developed method can be appropriate to apply pharmacokinetics and bioequivalence of clotiazepam.