A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated to quantify troxerutin in human plasma using rutin as the internal standard. After a simple protein precipitation with perchloric acid, the post-treatment samples were analyzed on a Phenomenex Synergi Fusion RP column interfaced with a tripe-quadrupole tandem mass spectrometer using positive electrospray ionization. The assay was linear over the concentration range 31.25-4 000 pg/mL using a 20 μL aliquot of plasma. The correlation coefficients for the calibration curves ranged from 0.999 5 to 0.999 6. The intra-and inter-day precisions over the entire concentration were not more than 13.5%. Acetonitrile and water (20∶80, v/v) is used as the isocratic mobile phase, with 0.1% of formic acid in water. The method was successfully applied for pharmacokinetic study after a single oral administration of 300 mg troxerutin to 18 healthy volunteers.