A rapid, sensitive, and selective ultra fast liquid chromatography-tandem mass spectrometry method was developed for quantitative determination of arenobufagin in rat plasma. Sample pretreatment involved a one-step protein precipitation with methanol using 0.1mL rat plasma. The separation was carried out on a Shim-pack XR-ODS II (75mm×2.0mm, i.d. 2.1μm) column with gradient elution at a flow rate of 0.30mLmin(-1). The mobile phase was acetonitrile and 0.1% formic acid in water. A post-column switching valve was applied to reduce the matrix effect. The detection was performed on a triple-quadruple tandem mass spectrometer in the multiple reaction monitoring mode after electrospray ionization. Linear calibration curves for arenobufagin were obtained over the concentration range 1.056-1056ngmL(-1), with a lower limit of quantification of 1.056ngmL(-1). The intra-day and inter-day precision values were lower than 15% and the accuracy ranged from 5.4% to 9.8% at all quality control levels. The method was successfully applied to the determination and pharmacokinetic study of arenobufagin in rat plasma following intraperitoneal administration.