A sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method for the determination of evodiamine and rutecarpine in rabbit plasma was developed and validated. The analytes and internal standard (IS) are extracted from plasma by one-step protein precipitation with acetonitrile, and separated on a Zorbax SB-C18 column (2.1 mm x 50 mm, 3.5 microm) using acetonitrile-0.1% formic acid as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode, and selective ion monitoring (SIM) mode used to quantify evodiamine and rutecarpine. The assay is linear over the range of 2-1600 ng/mL for evodiamine and rutecarpine, with a lower limit of quantification (LLOQ) of 5 ng/mL both for evodiamine and rutecarpine. Intra-day and inter-day precision are less than 12% and the accuracy are in the range of 90.9-104.3%. Furthermore, the newly developed method is successfully used for the determination of evodiamine and rutecarpine in rabbit plasma for pharmacokinetic study.