Styraxlignolide A is a pharmacologically active ingredient isolated from Styrax japonica Sieb. et Zucc. A rapid, selective, and sensitive liquid chromatographic method with electrospray ionization tandem mass spectrometry was developed for use in the quantification of styraxlignolide A in rat plasma. Styraxlignolide A was extracted from rat plasma using ethyl acetate at neutral pH. The analytes were separated on an Atlantis dC18 column using a mixture of methanol and ammonium formate (10 mM, pH 3.0) (70:30, v/v) and detected by tandem mass spectrometry in multiple reaction monitoring mode. The standard curve was linear (r(2) =0.9978) over the concentration range of 100-10000 ng/mL. The lower limit of quantification was 100 ng/mL using 50 μL of plasma sample. The coefficient of variation and relative error for intra- and inter-assays at four QC levels were 1.6-8.3% and from -12.0 to -1.7%, respectively. The present method was applied successfully to the pharmacokinetic study of styraxlignolide A after intravenous administration of styraxlignolide A at a dose of 10 mg/kg in male Sprague-Dawley rats.