Evodiamine and rutecarpine are two kinds of indole alkaloids contained in the fruit of Evodiae fructus, which have been shown to exhibit various bioactivities in humans. A liquid chromatography-tandem mass spectrometric method (LC-MS/MS) was developed for the determination of evodiamine and rutecarpine in human serum. The serum was extracted by solid-phase extraction (SPE) and analyzed using a C18 column and a mobile phase consisting of methanol-water (85:15) solution containing 5 mmol/L ammonium formate at a flow rate of 0.5 mL/min. The mass spectrometer was operated in positive mode, employing the extracted ion chromatogram (EIC) for detection and quantitation of evodiamine (m/z 288) and rutecarpine (m/z 304). Good linear relationships between the peak area and the concentration were obtained in the ranges of 5.2-1040 ng/mL and 10.2-1020 ng/mL, with correlation coefficients (r) of 0.999 and 0.998, for evodiamine and rutecarpine, respectively. The repeatabilities (RSD, n=6) of quantitation for evodiamine and rutecarpine were 2.18-4.00% and 2.99-5.67%, respectively, and the recovery ranged from 90.5% to 98.1%. A comparative study of the different ionization and quantitation modes, including ESI-MS, ESI-MS/MS, APCI-MS and APCI-MS/MS, was also accomplished. The MS/MS fragmentation mechanism of the base peak ([M+H](+), m/z 304) of evodiamine was investigated in order to identify the analytes in more complicated body fluid samples.