Abstract: OBJECTIVE To establish a high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of 34 kinds of prohibited sexual hormones in 3 different matrixes of cosmetics(water aqua, lotion and cream). METHODS Samples were ultrasonically extracted by solvent, and then purified by QuEChERS method with different kinds of salting out agent and purifying agent. The separation was performed on an Agilent ZORBAX SB C₁₈ column(2.1 mm×50 mm, 1.8 μm) with gradient elution using acetonitrile and water as the mobile phase. The determination was performed by MS/MS in multiple reaction monitoring(MRM) mode. Matrix matching calibration method was used for the quantification. RESULTS The results demonstrated that all 34 kinds of sexual hormones could be well separated. There were good linear relationships for the 34 compounds in the certain concentration ranges with their correlation coefficients (r²) larger than 0.99. The limits of detection and limits of quantification were in the range of 0.005-0.29 mg·kg^-1 and 0.02-0.95 mg·kg^-1, respectively. At 3 different concentrations, the recoveries of water aqua, lotion and cream samples were 88.3%-111.4%, 89.4%-114.6% and 86.9%-114.2%, respectively. And RSD were less than 10.0%. The method was used to test the samples, and progesterone was detected in two hair growth cosmetics. CONCLUSION The present method is accurate, efficient and easy pretreatment, and can be applied in the determination of the 34 sexual hormones in cosmetic products.