In this study, a new, simple, and reproducible high-performance liquid chromatographic method was developed for the determination of pioglitazone in human plasma. After liquid-liquid extraction with diethylether, samples were quantitated on a Nova-Pak C8 column using a mixture of acetonitrile-140mM K2HPO4 (40:60, v/v, pH = 4.45) as mobile phase with UV detection at 269 nm. The flow rate was set at 1.4 mL/min. Ethylparaben was used as internal standard and the total run time of analysis was approximately 7 min. The method was linear over the range of 25-1500 ng/mL of pioglitazone in plasma (r2 > 0.999). The within- and between-day precision values were in the range of 2.4-6.8%. The limit of quantitation of the method was 25 ng/mL. The method was successfully used to study the pharmacokinetics of pioglitazone in healthy volunteers.