This paper reports the validation of an assay for obtusifolin based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) and its application to a preclinical pharmacokinetic study in rats. After sample preparation of plasma and tissue homogenates by protein precipitation, the analyte and internal standard (IS) were separated by a reversed‐phase chromatographic system in a run time of 5.0 min and detected by negative ion electrospray ionization followed by selected reaction monitoring of the precursor‐to‐product ion transitions at m/z 283.0–268.1 for obtusifolin and m/z 329.0–314.1 for IS. The assay was linear in the concentration range 1.0–500 ng/ml with the LLOQ of 1.0 ng/ml. In the pharmacokinetic study of an intragastric administration of 1.3 mg/kg obtusifolin, the maximum plasma concentration of obtusifolin was 152.5 ± 62.3 ng/ml, reached at 0.39 ± 0.17 h. The AUC0–t and AUC0–∞ were 491.8 ± 256.7 and 501.7 ± 256.7 ng × h/ml, respectively, with an elimination half‐life of 3.1 ± 0.7 h. Obtusifolin was rapidly distributed into tissues, with the highest distribution in the liver and less in the brain. These results will give some insights for further pharmacological investigation of obtusifolin.