Abstract: OBJECTIVE The aim of this study was to develop and validate a LC-MS/MS method to simultaneously determination of Scutebarbatine A and Scutebarbatine B in rat plasma and applicated this method to pharmacokinetic study of Scutebarbatine A and Scutebarbatine B after oral administration of Scutellaria barbata extract. METHODS Plasma samples were prepared with ethyl acetate using liquid-liquid extraction. Then samples were separated by using BDS Hypersil C₁₈(50 mm×2.1 mm， 2.4 μm) with gradient elution program and oven temperature was set at 40 ℃. And the mobile phase was consisted with acetonitrile/water (0.05% formic acid) at a flow rate of 0.25 mL/min. Determination was carried out on a tandem mass spectrometer in positive ion mode using a multiple reaction monitoring (MRM) via a electrospray ionization (ESI) interface. After oral administration of 500 mg/kg Scutellaria barbata extract， plasma samples were collected and analyzed by using a DAS software to analyze to the pharmacokinetic parameters. RESULTS Scutebarbatine A and Scutebarbatine B were liner at the range of 0.512~258.0 ng/mL， 0.482~241.0 ng/mL， respectively with a r² larger than 0.994. Accuracy， precious， extraction efficiency， matrix effects and stability study were all meet the requirement of the bioanalytical method. The AUC_(0-t) for Scutebarbatine A and Scutebarbatine B were (88.69±12.4)μg/L·h， (57.09±9.84)μg/L·h， respectively. And the Cmax for Scutebarbatine A and Scutebarbatine B were (10.22±1.31)ng/mL and (6.27±0.80)ng/mL. CONCLUSION The method was simple， accurate， and sensitive， which could be used for the pharmacokinetic study of Scutebarbatine A and Scutebarbatine B after oral administration of Scutellaria barbata extract.