An accurate and sensitive analytical method has been developed for the quantification of genistein in dog plasma using high-performance liquid chromatography/tandem mass spectrometry. Genistein and diclofenac (internal standard) were extracted from the plasma sample using methyl tert-butyl ether and then separated on an Agilent Zorbax C18 column using a gradient mobile phase. The detector was a Q-trap mass spectrometer with an electrospray ionization interface operating in the multiple reaction monitoring mode. The assay was linear over the concentration range of 0.10-500 ng/mL with a lower limit of quantification of 0.10 ng/mL. The method was shown to be reproducible and reliable, with inter-day and intra-day accuracy and precision within ±15%. The method was successfully applied to a pharmacokinetic comparison of immediate and extended release tablets in beagle dogs after oral administration. Immediate release tablets showed rapid genistein absorption, with mean peak concentration of 726 ± 199 ng/mL reached at 0.2 ± 0.0 h. However, the absorption of genistein was considerably slower and more sustainable for extended release tablets. The relative bioavailability of the extended release tablet over the immediate release formulation was estimated to be 134 ± 47% based on the AUCInf values from non-compartmental analysis.