The invention discloses a UPLC/MS/MS detection method of the concentration of 4'-hydroxydiclofenac in a liver microsome. The method comprises the following steps: 1, establishing a liver microsome incubation system, and adding an internal standard substance and a series of concentrations of a 4'-hydroxydiclofenac reference substance to prepare a standard curve sample; 2, injecting the standard curve sample to an UPLC/MS/MS instrument, detecting, and obtaining a 4'-hydroxydiclofenac standard curve according to the above obtained detection result; and 3, preparing a sample to be detected according to a technology in step 1, injecting the sample to be detected to the UPLC/MS/MS instrument, detecting under conditions same to those in step 2, and obtaining the concentration of 4'-hydroxydiclofenac in the sample to be detected according to the standard curve. The detection method has the advantages of short analysis time, high sensitivity, small sample introduction amount, methodology verifying requirement meeting, suitableness for determining the concentration of 4'-hydroxydiclofenac in the liver microsome incubation sample, and wide application prospect.