Objective: Pre-clinical safety/toxicity studies are routinely conducted in rodent and non-rodent species. Often, drug metabolizing enzymes activities are determined to assess drug candidate induction potential and gain understanding of pharmacokinetic properties following repeat dosing. Drugs intended for animal use may undergo similar testing but advantageously can be conducted in target species. In vitro enzyme activity assays are well-established for mouse, rat, dog and monkey, but only limited information on the cytochrome P450 (CYP) and uridine 5’-diphosphoglucuronosyl transferase (UGT) activities is available for the domestic shorthair cat. The purpose of this investigation was to develop CYP and UGT enzyme activity assays in cat liver microsomes to assess drug-induced changes ex-vivo. Methods: Male (n=3) and female (n=3) cat livers were homogenized and microsomes prepared by differential centrifugation. Male and female pools were then characterized for various CYP enzyme activities generally considered selective for the major xenobiotic metabolizing CYP families (e.g. CYP1, 2, 3 and 4) in mammals. Activities and target enzyme examined were 7-ethoxyresorufin o-deethylase/CYP1A; 7-benzyloxyresorufin o-dearylase/CYP2B; lauric acid 11- and 12-hydroxylase/CYP2E and CYP4, respectively and testosterone 6β-hydroxylase/CYP3A. In addition, 4-methylumbelliferone (4-MU) - and p-nitrophenol glucuronosyl transferase activities were assessed. Results: In general, kinetic parameters were not remarkably different compared to other preclinical species examined in this laboratory. It is generally believed that the cat has little or no capacity to glucuronidate drugs. Thus it was somewhat surprising to find that 4-MU was glucuronidated albeit at generally lower activity compared to other preclinical species. p-Nitrophenol kinetic parameters were not determined because catalytic activity was not detected. Conclusions: Liver microsomal CYP and UGT enzyme activity assays were developed in domestic cat and are suitable for assessing changes in enzyme levels both male and female animals.