A selective, sensitive, and accurate LC-MS/MS method for the simultaneous determination of isoalantolactone and alantolactone in rat plasma has been developed using psoralen as the internal standard. LC-MS/MS analysis was carried out on a Triple Quadrupole mass spectrometer using positive ion ESI and the selected reaction monitoring mode. The assays were linear in the range of 7.5-750 ng/mL for isoalantolactone and 5.5-550 ng/mL for alantolactone. The average recoveries in plasma samples both were better than 85%. The intra- and inter-day precision and accuracy values were found to be within the assay variability criteria limits according to the US FDA guidelines. The method was successfully applied to pharmacokinetic studies of the two structural isomers after an intravenous injection of Inula helenium formulation to rats.