Diet-induced obese (DIO) mice have been commonly used extensively as an animal model of obesity and diabetes in the efficacy assessment for new drug candidates. Physiological and biochemical alterations are reported in DIO mice due to modulations of drug-metabolizing enzymes and high calorie intake. Limited studies have been reported regarding the effect of obesity/diabetes on pharmacokinetics (PK) in animals. A simple, specific and sensitive rapid LC-ESI-MS/MS method has been developed and validated for the quantification of chrysin (5,7-dihydroxyflavone) using tolbutamide as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a simple protein precipitation. Chromatographic separation of 5,7-dihydroxyflavone and IS was performed on Atlantis C-18 column using an isocratic mobile phase comprising 0.2% formic acid in water and acetonitrile (20:80, v/v) at a flow rate of 0.9 mL/min. Elution of 5,7-dihydroxyflavone and IS occurred at ~2.49 and 2.34 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 4.50-4500 ng/mL. This novel method has been applied to a pharmacokinetic study in lean and DIO mice.