A rapid and sensitive method for the determination of pinostrobin in rat plasma was developed using liquid chromatography tandem mass spectrometry (LC–MS/MS) for the first time. Isoliquiritigenin was used as an internal standard in rat plasma. Chromatographic separation was performed on an HiQ Sil C18 column with isocratic elution at a flow rate of 1 mL/min. The mobile phase consisted of water and methanol (9:91, v/v) containing 0.1% formic acid. The quantification limit was 10 ng/mL within a linear range of 10–1000 ng/mL (R = 0.9984). The intra- and inter-day assay precision ranged from 3.8–5.3% to 3.2–5.2%, respectively, and the intra- and inter-day assay accuracy was between 93.2–95.1% and 95.5–104.3%, respectively. Our results indicated that the LC–MS/MS method is effective for pharmacokinetic study of pinostrobin in rat plasma.