This study aimed to develop and validate a simple and sensitive ultra high performance liquid chromatography tandem mass spectrometry method for the simultaneous determination of sophoraflavanone G and kurarinone in rat plasma by using rutin as the internal standard. Then, the developed method was applied to investigate the pharmacokinetics of sophoraflavanone G and kurarinone in rats after dosing the flavonoid extract from Sophora flavescens. Plasma samples were processed using a liquid-liquid extraction procedure with ethyl acetate. The analysis was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring with an electrospray ionization source in negative ionization mode. Quantitative ion transitions of m/z 423.2→161.2, 437.2→161.1, and 609.3→300.3 were monitored for sophoraflavanone G, kurarinone, and rutin, respectively. The calibration curves of the two analytes exhibited good linearity (r >0.9923) over the range of 0.1-200 ng/mL for sophoraflavanone G and 0.1-1000 ng/mL for kurarinone. Relative standard deviations were less than 13.2% for the intra- and inter-day precisions and no more than 12.6% for the recovery, showing good precision and satisfactory accuracy of the developed method. The validated method was successfully applied to the pharmacokinetic study of sophoraflavanone G and kurarinone after a single intravenous (25 mg/kg) and oral (500 mg/kg) administration of the flavonoid extract from S. flavescens, and the absolute bioavailability for sophoraflavanone G and kurarinone was about 36 and 17%, respectively.