Abstract: A method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of flufenacet in rat tissues and the investigation of the tissues distribution behavior of test item in rats after intragastric administration. Rat tissue homogenate was extracted withacetonitrile and analyzed by LC-MS/MS. Chromatographic separation was performed on a Phenomenex reverse C₁₈ (50 mm × 4.6 mm, 5 μm) column at a flow rate of 0.6 mL/min with iscratic elution. The mobile phase was 72% acetonitrile/water solution (containing 0.1% formic acid). Electrospray ionization (ESI) with a positive-ion and multiple reaction monitoring mode was used. Quantitative ion pair was 364.1/194.2. The linear ranges of flufenacet in heart, liver, spleen, lung, kidney, brain, muscle, testis and fat were all ranged from 0.002 to 0.1 mg/L. The linear ranges of flufenacet in large intestine, small intestine and stomach were in the range of 0.10-10 mg/L. The correlation coefficient between the mass concentration of flufenacet and the corresponding peak area was good (r > 0.99). The precision of this method for QC samples was less than 11%. The matrix effect was in the range of 94.5%-95.6%. The recovery was in 96 %-107%, and the stability was in 95%-104%. All these parameters met the requirements of methodology. The experimental results of tissue distribution of flufenacet showed that flufenacet was widely and rapidly distributed in rats after the gavage of 400 mg/kg flufenacet. Flufenacet mainly distributed in large intestine, small intestine, stomach and lung. And after the concentration of each tissue reached the peak, flufenacet could be eliminated in all tissues. An efficient, specific and sensitive LC-MS/MS method was established, and this validated method could be used in the analysis of flufenacet in rat tissues to realize the tissue distribution behaviors.