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For the first time, an HPLC method was developed and validated for the determination of stemoninine in plasma after oral and intravenous administration of the extract of the roots of Stemona tuberosa to rats. Plasma samples were analyzed on a Waters reversed-phase C-18 column using a gradient mobile-phase of eluent A (water containing 0.1% formic acid and 0.2% triethylamine, pH3.68) and eluent B (acetonitrile-water, 50: 50, v/v). The flow rate was 1.0 mL/min and the detector wavelength was 210 nm. The Waters Oasis solid-phase extraction cartridge was applied for the preparation of plasma samples with high recovery. A good linear relationship was obtained in the concentration range of 1.55-124 mu g/mL (r = 0.9995). The limits of quantification and detection were 1.55 and 0.42 mu g/mL, respectively. The average recoveries ranged from 91.11 to 96.43% in plasma at stemoninine concentrations of 3.10, 62.0 and 99.2 mu g/mL. Intra-and inter-batch coefficient of variations were 3.27-5.37% and 2.49-3.92%, respectively. This method was successfully applied to pharmacokinetic studies after oral and intravenous administration of Stemona tuberosa extract in rats. Copyright (c) 2010 John Wiley & Sons, Ltd.