Abstract: Parthenolide is a sesquiterpene lactone derived from the plant feverfew (Tanacetum parthenium) that possesses multiple anti-inflammatory and anti-cancer properties. A specific, sensitive, accurate and precise LC-MS/MS method was developed and validated in the quantitative analysis of parthenolide in rat plasma. A liquid-liquid extraction method was used to separate the analyte and the internal standard (IS, costunolide) from plasma. A water-methanol mobile phase system was utilized in the gradient chromatographic separation. The calibration curve with good linearity (r²>0.99) was established between 2 and 128 ng·mL^-1 with accuracy and precision within acceptable limits at different QC levels. High extraction recovery was achieved for both parthenolide (89.55%-95.79%) and IS (96.87%). Based on this LC-MS/MS method, the plasma stability and pharmacokinetics of parthenolide were assessed in rats. Parthenolide was proved to be very unstable in rat plasma, and was distributed and eliminated quickly in vivo, with a half-life less than 90 min. A high dose of parthenolide (80 mg·kg^-1) resulted in a very low initial concentration (138.86±21.07 ng·mL^-1). The systemic exposure of parthenolide (area under the curve) increased disproportionally from 40 mg·kg^-1 dose group to 80 mg·kg^-1 dose group. The present study may provide helpful information for the development of parthenolide as a drug candidate.