<p>Background: A simple and sensitive high performance liquid chromatography-electrospray ionization mass spectrometry method has been evaluated for the assignment of clonidine hydrochloride in human plasma.<br /> Methods: The mobile phase composed of acetonitrile-water 60:40 (<em>v/v</em>) and 0.2% formic acid 20<em> &micro;l</em> of sample was chromatographically analyzed using a repacked ZORBAX-XDB-ODS C₁₈ column (2.1 <em>mm</em>x30 <em>mm</em>, 3.5 <em>&mu;</em>). Detection of analytes was achieved by tandem mass spectrometry with Electrospray Ionization (ESI) interface in positive ion mode operated under the multiple-reaction monitoring mode (m/z 230.0 &rarr;213). Sample pretreatment consisted of a one-step Protein Precipitation (PPT) with methanol and perchloric acid (HClO₄) of 0.10 <em>ml</em> plasma.<br /> Results: Standard curve was linear (r=0.998) over the concentration range of 0.01-10.0 <em>ng/ml</em> and showed suitable accuracy and precision. The Limit of Quantification (LOQ) was 0.01 <em>ng/ml</em>. The mean (SD) Cmax, Tmax, AUC_0&ndash;tand AUC_{0&ndash;&infin;} values after administration of the test and reference formulations, respectively, were in this manner: 6.16 (0.32) versus 6.21 (0.07) <em>ng/ml</em>, 30.12 (0.86) versus 30.13 (0.73) <em>hr</em>, 290.37 (1.13) versus 293.39 (1.22) <em>ng/ml/hr</em>, and 350.17 (1.98) versus 352.96 (1.67) <em>ng/ml/hr</em>. The mean (SD) t1/2 was 120.12 (1.90) <em>hr</em> for the test formulation and 120.96 (1.54) hr for the reference formulation. No statistical differences were showed for Cmax and the area under the plasma concentration-time curve for test and reference tablets.<br /> Conclusion: The method is rapid, simple, very steady and precise for the separation, assignment, pharmacokinetic and bioavailability evaluation of clonidine in healthy Iranian adult male volunteers.</p>