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Objective Alkyne hormones is a kind of synthetic steroids substances with low molecular weight, strong lipophilicity and good biological activites, which play an important role in the regulation of humans physiological and metabolic process.Recently, A novel quantitative method using liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed for simultaneous determination of Ethinylestradiol, Quinestrol, Norethisterone, Levonorgestrel, Danazol, Nilestriol, Norethisterene Acetate, Etaynodiol Diacetate, Norethisterone enanthate in human serum.Methods After precipitation by 10% potassium ferrocyanide 20% zinc acetate,the samples were combined with azido compound by click reaction.Then, the reaction products were detected by liquid chromatography-triple quadrupoles mass spectrometry (LC-QqQ) using multiple reaction monitoring (MRM) in the negative scan mode.Chromatographic separation of the target analytes from the sample extracts was achieved with a reversed-phase Aglient zorbax C18 coluum (150 mm × 4.6 mm i.d., 5 μm), with a binary mobile phase composed of 5mM NH4COOH in water (eluent A) and acetonitrile (eluent B) pumped at a flow rate of 0.30 mL/min.The run time was 10 min and the injection volume was 10 μL.Capillary voltage for positive electrospray was set at-4.5 kV, with a turbo ion spray interface source temperature at 500 ℃.Declustering potential (DP), collision energy (CE) and collision cell exit potentials (CXP) were optimized at-108 V,-30 eV and-15 V, respectively.A 200-ms dwell time with a 5-ms pause time was used.Results and conclusion The azido derivatives of analyte obtained by the combination ofazide compound and analytes in the click reaction, with larger molecular weight, improved the ionization efficiency of the analytes in the electrospray ionization source and also reduced the interference of impurity ions in the matrix.Besides, the product of click reaction is available for the quantitative determination with high yield, with less by-products, easy separation and simple reaction condition.The linear ranges of nine alkyne hormones were validated from 1.0 ng/mL to 200 ng/mL in plasma and the limits of detection varies between 0.1 and 7.5 ng/mL, respectively.The established LC-MS/MS method is sensitive, accurate, simple and suitable for the analysis of Ethinylestradiol, quinestrol, norethisterone, levonorgestrel, danazol, nilestriol, norethisterene acetate,etnynodiol diacetate, norethisterone enanthate in human serum.