Abstract: Background and Objective: N-acetyltransferases (NATs) were phase II drug-metabolizing enzymes, NATs activity was associated with certain adverse drug reactions. This study aimed to establish a method for NAT1 and NAT2 activity assay by using living human hepatoma HepaRG cells and tried to apply the established method for preliminary screening of the potential inhibitors of NAT1 and NAT2. Materials and Methods: Immunohistochemistry and western-blot techniques were used to evaluate the expression of NAT1 and NAT2 in HepaRG cells. Non-cytotoxic concentration of the specific substrates of NAT1 and NAT2, i.e., 4-aminosalicylic acid and isoniazid were incubated with HepaRG cells for 8 h, the culture medium was collected and determined by LC-MS/MS to evaluate the activity of NAT1 and NAT2. The effects of 4-aminosalicylic acid and isoniazid on NAT1 and NAT2 expression in HepaRG cells were also tested by western-blot and the reported NAT inhibitors were also used here to evaluate the sensitivity of the established method for NAT activity assay. Results: The results showed that NAT1 and NAT2 were expressed in HepaRG cells and NAT1 and NAT2 activity could be evaluated by quantifying the acetylated metabolites of their specific substrate 4-aminosalicylic acid and isoniazid, respectively. NAT activity detected by using living HepaRG cells was in parallel with that from traditional method for NAT activity assay, i.e., reactions using cells lysate. NAT activity assay by using living HepaRG cells was suitable for high-throughput screening of the potential inhibitors of NAT1 and NAT2, indicating the validity of the established method for NAT activity assay. Conclusion: The present study established a low-costing and stable method for screening of compounds with NAT1 and NAT2 inhibitory properties, which was helpful for new drug discovery and development.