To establish an LC–MS/MS method for determination of isorhamnetin-3-O-neohesperidoside and investigate its application on pharmacokinetic study in rats. Eight rats were given 5 mg·kg −1 isorhamnetin-3-O-neohesperidoside after intravenous administration. A highly sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of isorhamnetin-3-O-neohesperidosidein rat plasma using rutin as internal standard. The analytes and rutin (internal standard) were extracted with methanol followed by a rapid isocratic elution with 10 mmol·L −1 ammonium acetate buffer/methanol (20 : 80, V/V) on a C 18 column (150 mm × 2.1 mm, I.D., 5 μm) and subsequent analysis by mass spectrometry in the multi-eaction-monitoring mode. The assays were linear over the concentration range of 0.01–10 μg·mL −1 for isorhamnetin-3-O-neohesperidosidein rat plasma. The lower limit of quantifications for isorhamnetin-3-O-neohesperidoside was 0.01 μg·mL −1 . The validated method is successfully applied to determine the plasma concentrations of isorhamnetin-3-O-neohesperidosidein in rats.