Formononetin-7-O-beta-d-glucoside has been proved to have significant anti-inflammatory effect. To evaluate its rat pharmacokinetics, a rapid, sensitive, and specific liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantification of formononetin-7-O-beta-d-glucoside and its main metabolite formononetin in rat plasma. Samples were pretreated using a simple protein precipitation and the chromatographic separation was performed on a C-18 column by a gradient elution using a mobile phase consisting of water and acetonitrile both containing 0.1% formic acid. Both analytes were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. The assay showed wide linear dynamic ranges of both 0.10-100 ng/mL, with acceptable intra- and inter-batch accuracy and precision. The lower limits of quantification were both 0.10 ng/mL using 50 mu L of rat plasma for two analytes. The method has been successfully used to investigate the oral pharmacokinetic profiles of both analytes in rats. After oral administration of formononetin-7-O-beta-d-glucoside at the dose of 50 mg/kg, it was rapidly absorbed in vivo and metabolized to its metabolite formononetin. The plasma concentration-time profiles both showed double-peak phenomena, which would be attributed to the strong enterohepatic circulation of formononetin-7-O-beta-d-glucoside.