The invention discloses a UPLC/MS/MS detection method of the 6 alpha-hydroxypaclitaxel concentration in a liver microsome. The UPLC/MS/MS detection method comprises the following steps that 1, a liver microsome incubation system is established, internal standard substance and serial concentrations of 6 alpha-hydroxypaclitaxel comparison products are added to prepare a standard curve sample; 2, the standard curve sample is injected into a UPLC/MS/MS liquid chromatograph/mass spectrometer for detection, and a standard 6 alpha-hydroxypaclitaxel curve is obtained according to a detection result; 3, a sample to be detected is prepared by adopting the same method as the step 1 and is injected into the UPLC/MS/MS liquid chromatograph/mass spectrometer, detection is performed under the same conditions as the step 2, and the 6 alpha-hydroxypaclitaxel concentration in the sample to be detected is obtained according to the standard curve. The UPLC/MS/MS detection method is short in analysis time, high in sensitivity and small in sample size, meets the methodological validation requirement, is suitable for determination of the 6 alpha-hydroxypaclitaxel concentration in the liver microsome incubation sample and has wide application prospect.