A sensitive, rapid and specific liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed and validated for the determination of aristolochic acid-I (AA-I) in rat plasma. Finasteride was used as the internal standard (IS). The analyte was separated on a Zorbax Eclipse XDB-C-18. column by isocratic elution with methanol-10 mM ammonium acetate (75:25, v/v, pH = 7.3) at a flow rate of 0.25 mL/min, and analyzed by mass spectrometry in positive multiple reaction monitoring mode. The precursor-to-product ion transitions of m/z 359.0 -> 298.2 and m/z 373.1 -> 305.2 were used to detect AA-I and IS, respectively. Good linearity was achieved over a range of 0.4-600 ng/mL. Intra- and inter-day precisions measured as relative standard deviation were less than 13.5%, and accuracy ranged from 94.2 to 97.5%. The developed method was successfully applied in the pharmacokinetic study of AA-I in rats. Copyright (C) 2009 John Wiley & Sons, Ltd.