A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6x50 mm, 5.0 microm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5-100 ng/mL with a correlation coefficient (r) of > or = 0.996. The intra- and inter-day assay precision ranged from 1.66-6.82% and 1.87-6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98-107.56% and 90.54-105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC-MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.