Abstract: Aim To develop a sensitive and specific HPLC method for determination of trigonelline in rabbit plasma, and study the pharmacokinetics in rabbit. Methods After ig of fenugreek extract and iv of trigonelline in rabbit, the biological samples could be well purified after precipitation of protein with methanol and acetonitrile. Asahipak NH₂P-50 column was used,the mobile phase consisted of acetonitrile-water (90∶10) at a flow-rate of 1.2 mL·min^-1, and detection wavelength was set at UV 265 nm. The column temperature is 30 ℃. Results The calibration curve was linear in the range from 0.98 mg·L^-1 to 31.28 mg·L^-1, with R=0.9986, the detection limit of this method was 50 μg·L^-1. The concentration-time curves of trigonelline in rabbits after ig and iv administration were shown to fit one-compartment and two-compartment open model, respectively. The main parameters after ig of fenugreek extract were as follows: T_1/2(Ka) was 0.9 h, T_1/2(Ke) was 2.2 h, V was 0.64 L·kg^-1, AUC was 1.93 mg·min·L^-1. The main parameters after iv of trigonelline were as follows: T_1/2α was 10.8 min, T_1/2β was 44.0 min, K₂₁ was 0.044 min^-1, K₁₀ was 0.026 min^-1, K₁₂ was 0.017 min^-1, AUC was 931.0 mg·min·L^-1. Conclusion Trigonelline showed a middle rate of absorption and fast rate of elimination in rabbit. Meanwhile, the method is simple,accurate,with a good reproducibility, and it provide a basic method for the investigation of trigonelline and fenugreek pharmacokinetics.