Abstract: A method for ultra performance liquid chromatography-tandem mass spectrometric(UPLC-MS/MS) was developed for the simultaneous identification and determination of 6 sex hormones in the duck liver. Components were extracted from homogenized sample with Methyl Tertiary Butyl Ether(MTBE) ordinally, cleaned by Sep-Pak Silica SPE column, and detected by electrospray ionization mass spectrometry in the positive ion mode. This method is more suitable for determination of sex hormones in animal foods.