A rapid and sensitive method for determination of ascaridole in rat plasma was developed based on gas chromatography-mass spectrometry (GC/MS). The analyte and internal standard (IS), naphthalene, were extracted from plasma with ethyl acetate and then separated by GC on a HP-5MS capillary analytical column (30 m x 0.25 mm, 0.25 microm) and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode (SIM). Excellent linearity was found to be from 10 to 1,000 ng/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL. The accuracy was between 85.3% and 114.0%, and the precision was less than 14.5% (intra- and inter-day). The method was successfully applied to investigate the pharmacokinetic study of ascaridole in rats after a single oral dose of 30, 60 and 120 mg/kg, respectively.