Cajaninstilbene acid (CSA; 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid) is a major active constituent of pigeonpea leaves, has been proven to be effective in clinical treatment of diabetes, hepatitis, measles and dysentery. A rapid and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of CSA in rat plasma and various tissues (brain, heart, lung, liver, spleen, small intestine and kidney) of rat for the first time. Rat plasma and tissue distribution pre-treated by protein precipitation with acetoacetate was analyzed using LC-MS/MS with an electrospray ionization (ESI) interface, and isoliquiritigenin was used as an internal standard. Chromatographic separation was achieved on a HIQ Sil C(18) column with the mobile phase of water and methanol (9:91, v/v) containing 0.1% formic acid and resulted in a total run time of 10 min. The isocratic elution mode pumped at a flow rate of 1.0 mL/min. The lower limit of quantification (LLOQ) which was 10 ng/mL. The calibration curve was linear from 10 to 6000 ng/mL (R=0.9967) for plasma samples and 10 to 6000 ng/mL (R>or=0.9974) for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 0.6% to 6.1% and 1.5% to 6.6%, respectively, and the intra- and inter-day assay accuracy was 93.5-104.6% and 93.3-107.5%, respectively. Recoveries in plasma and tissues ranged from 95.0% to 106.8%. The method was successfully applied in pharmacokinetic and tissue distribution studies of CSA after oral administration to rats. The pharmacokinetics of CSA showed rapid absorption and elimination (T(max), 10.7+/-0.31 min; t(1/2), 51.40+/-6.54 min). After oral administration in rats, CSA was rapidly and widely distributed in tissues. High concentrations were found in liver and kidney indicating that CSA was possibly absorbed by liver and eliminated by kidney.