The invention discloses a UPLC/MS/MS detection method for acetaminophen concentration in a liver microsome. The detection method comprises the following steps: (1) building a liver microsome incubation system, adding an internal standard substance and acetaminophen reference substances of series concentrations, and preparing a standard curve sample; (2) injecting the standard curve sample into a UPLC/MS/MS liquid-mass chromatography for detection, and obtaining a standard acetaminophen curve according to the detection result; (3) preparing a sample to-be-detected according to a method the same as the step (1), injecting the sample to-be-detected into the UPLC/MS/MS liquid-mass chromatography, carrying out detection under the condition the same as that of the step (2), and obtaining the acetaminophen concentration of the sample to-be-detected according to the standard curve. The detection method provided by the invention has the advantages that the analysis time is short; the sensitivity is high; the sample volume is small; the requirements of various methodology validation are met; the method is suitable for detection of acetaminophen in a liver microsome incubation sample; the application prospect is broad.