The invention discloses a UPLC/MS/MS (ultra-performance liquid chromatography/tandem mass spectrometry) detection method for concentration of 6 beta-hydroxytestosterone in liver microsome. The method comprises steps as follows: (1), an incubation system for the liver microsome is established, an internal standard substance and a 6 beta-hydroxytestosterone reference substance with serial concentration are added, and a standard curve sample is prepared; (2), the standard curve sample is injected into a UPLC/MS/MS liquid chromatograph/mass spectrometer for detection, and a standard curve of 6 beta-hydroxytestosterone is obtained according to a detection result; (3), a to-be-detected sample is prepared with the same method in the step (1), injected to the UPLC/MS/MS liquid chromatograph/mass spectrometer and detected under the same conditions in the step (2), and the concentration of 6 beta-hydroxytestosterone in the to-be-detected sample is obtained according to the standard curve. According to the detection method, the analysis time is short, the sensitivity is high, the sample amount is small, and the method meets requirements of methodology validation, is suitable for measurement of the concentration of 6 beta-hydroxytestosterone in a liver microsome incubation sample and has a wide application prospect.