A simple and sensitive high-performance liquid chromatographic method was developed for the simultaneous determination and pharmacokinetic analysis of seven alkaloids dehydroevodiamine (DHED), 10-hydroxyrutaecarpine (HDR), evodiamine (EDM), rutaecarpine (RCP), 1-methyl-2-n-nonyl-4(1H)quinolone (MNQ), evocarpine (ECP), and dihydroevocarpine (DHE), and two flavonoids isorhamnetin-7-O-rutinoside (RIM) and diosmetin-7-O-β-d-glucopyranoside (GRD) in rat plasma after oral administration of Wuzhuyu decoction. The flow rate was kept at 1.0 ml/min and the detection wavelength was set at 300 nm. The calibration curves were linear in the range of 0.5013-30.076 μg/ml for DHED, 0.2161-21.608 μg/ml for RIM, 0.161-12.876 μg/ml for HDR, 0.2146-21.457 μg/ml for GRD, 2.0464-40.928 μg/ml for EDM, 1.0398-31.194 μg/ml for RCP, 0.5970-35.818 μg/ml for MNQ, 0.8371-20.928 μg/ml for ECP, and 0.5167-31.003 μg/ml for DHE. The precision (relative standard deviation (RSD), %) for all was less than 10% and the accuracy (relative error (RE), %) was within ± 10%. The results demonstrated that the assay had remarkable reproducibility with acceptable accuracy and precision. The lower limit of quantifications for the compounds in plasma ranged from 0.12 to 0.23 μg/ml and the lower limit of detections ranged from 0.024 to 0.076 μg/ml. This validated method has been successfully applied in the pharmacokinetics study of seven alkaloids and two flavonoids after orally administrating the Wuzhuyu decoction to rats.