Pseudolaric acid B (PAB), which is the main biologically active diterpene acid of Pseudolarix kaempferi, has presented anti-fungal, anti-tumor, anti-fertility, and anti-tubulin activities. In this study, a sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method with multiple-reaction monitoring mode was employed for quantification of PAB in rat plasma. The calibration curve was linear over the range of 0.86-288 ng/mL with correlation coefficient (r) greater than 0.995 for PAB, and the lower limit of quantification was 0.86 ng/mL in rat plasma. The accuracy of PAB was between -9.1% and 7.0% relative error, and precision ranged from 1.2 to 10.6% relative standard deviation. This method was successfully applied to the pharmacokinetic studies of PAB in Sprague-Dawley rats. After single intravenous administration of 2.0, 4.0, and 8.0mg/kg PAB to rats, the t1/2 were (16.1 ± 5.6), (30.0 ± 13.7), and (27.4 ± 5.3)min, respectively. The pharmacokinetics (C(2min), AUC) of PAB within the used dosage range were in accordance with linear pharmacokinetic characteristics.