Objective To develop a determination method for streptomycin and dihydrostreptomycin in cheese using high performance liquid chromatography-tandem mass spectrometry.Methods The samples were extracted with 20 mmol/L Na₂HPO₄ solution （pH 7.4， adjusted by 6 mol/L HCl） through sonication. The purification and enrichment were carried out by a WCX mixed-mode weak cation-exchange SPE column. The samples were separated by UPLC， ionized by electrospray ionization triple quadrupole mass spectrometer and detected under selected reaction monitoring mode.Results The linear range was 1-200 ng/mL with correlation coefficients （r） higher than 0.996. The spiked recoveries ranged from 82.2% to 116.2% with relative standard deviations of 2.67%-7.2%. The limits of detection of streptomycin and dihydrostreptomycin were both 5 μg/kg and the limits of quantitation were both 10 μg/kg.Conclusion This method is rapid， simple， highly sensitive and suitable for the detection of streptomycin and dihydrostreptomycin in cheese.