Abstract: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of trace levels of diethylstilbestrol, hexestrol and dienestrol in pork liver. The sensitivity was achieved by using ammonium fluoride as an eluent additive, which significantly enhanced the analytical signals on average about 10 times. The analytes were extracted with acetonitrile containing 1%（V/V） acetic acid by ultrasonic extraction. Then, the extracts were purified by dispersive solid phase extraction with C18 and then diluted with water for LC-MS/MS analysis. Chromatographic analysis was conducted on a Waters Acquity UPLC HSS T3 column using acetonitrile-0.1 mmol/L ammonium fluoride as mobile phase with gradient elution. The analytes were detected by negative electrospray ionization-tandem mass spectrometry under multiple reaction monitoring mode. The enzymolysis method was investigated using β-glucuronidase/sulfatase enzyme from Helix pomatia for 12 h at 37 ℃. The results showed the cis-trans-tautomerism of diethylstilbestrol and diethylstilbestrol-d8 during enzymolysis, and the signals of the two compounds were reduced significantly by 60%. Only the same isotope internal standard calibration was used to obtain the accurate results with enzymolysis method.The quantification was achieved using external standard method without enzymolysis. Because the samples turned out to be sufficiently dilute, the matrix effects were soft (enhancement of 9.6%-13.6%) and negligible. The recoveries at fortification levels of 0.4, 0.8, 4.0 μg/kg in pork liver ranged from 89.5% to 109.4% with the relative standard deviations of 2.2%-7.8%. The limits of quantification (S/N>10) were 0.4 μg/kg for all analytes. The method does not require concentration for the whole sample processing, and need not employ isotope internal standard or matrix-matched calibration for quantification. It is also highly simple, reliable and sensitive in detecting the trace diethylstilbestrol, hexestrol and dienestrol in pork liver.