An ex vivo assay determining the flavin-containing monooxygenase (FMO) activity in perfused rat liver has been developed by assessing the rate of thiobenzamide S-oxide (TBSO) formation from the infused thiobenzamide (TB). The hepatotoxicity by TB or TBSO was not a critical factor for maintaining the FMO activity for up to 50 min. The FMO activity expressed in nmoles TBSO produced/g liver/min was the same for the recycling and non-recycling perfusion. This implies that reduction of the oxidized TBSO back to the parent compound (TB) is negligible. Hydrolysis of the collected perfusates with either β- glucuronidase or arylsulfatase did not increase the TBSO level and thus, TBSO does not appear to undergo conjugation either to glucuronide or sulfate esters. Thus, measuring the rate of TB S-oxidation in the isolated perfused liver with 1 mM TB for 50 min provides a useful tool for evaluation of the hepatic FMO activity in the absence of hepatic necrosis and without the interferences caused by further conjugation or back reduction of the TBSO to the parent TB.