In this study we probe the use of 7‐Dimethylamino‐4‐trifluromethylcoumarin (Coumarin 152, C152) as a fluorogenic substrate for in vitro studies of drug metabolism in human liver microsomes (HLM). Examining spectral properties of the formed fluorescent products and using LC‐MS/MS for monitoring C152 metabolism we demonstrate sequential formation of two N‐demethylated products – 7‐Methylamino‐4‐trifluoromethylcoumarin (desmethyl‐Coumarin 152, DC152) and amino‐4‐trifluoromethylcoumarin (Coumarin 151, C151). The predominant product formed by HLM is DC152, which accounts for >70% of C152 metabolized. Using insect cell microsomes containing recombinant P450 enzymes (Supersomes™) we found that C152 is metabolized by CYP2B6, CYP3A4, CYP2C19, CYP1A2 and CYP2C9 listed in the order of decreasing rates of turnover. The KM values exhibited by CYP2B6, CYP3A4, CYP1A2 and CYP2C19 were of the same order of magnitude (1.8 – 4.8 μM). Importantly, the major metabolizers of C152 – CYP2B6, CYP3A4, CYP2C19 and CYP1A2 – demonstrated different ability to catalyze the second demethylation step, with CYP1A2 being the most potent catalyst in this reaction. While over half of the substrate metabolized by CYP1A2 underwent the second demethylation, the fraction of C151 formed by other enzymes did not exceed 30%, similar to what is observed in HLM. Although the rate of turnover of C152 with CYP2B6 is higher than that observed with CYP3A4, the latter appears to be the major metabolizer of C152 due to its high abundance in HLM. This is confirmed by ketoconazole (KCZ) inhibition experiments. We demonstrate that in both CYP3A4 Supersomes™ and HLM over 80% of C152 turnover is suppressed by KCZ, whereas the activities of CYP1A2, CYP2B4 and CYP2C19 with C152 remain largely unaffected. Our results demonstrate that C152 can be used for probing functional interrelationships between multiple cytochrome P450 species in HLM as an example of a substrate that is almost exclusively metabolized by CYP3A4 though having similar affinities to most major human drug‐metabolizing cytochromes P450. In addition, we establish an easy and reliable fluorimetric assay for monitoring and analyzing the metabolism of C152 that allows discriminating between the two sequential steps of its N‐demethylation.Support or Funding InformationThis research was supported by the National Institute On Alcohol Abuse And Alcoholism of NlH under Award Number R21AA024548