Uridine 5′-diphospho-N-acetylgalactosamine glycosyltransferases (UGTs) and sulfotransferases (SULTs) are 2 phase II enzymes that are actively involved in detoxification processes as well as in drug metabolism. Compared with cytochrome P450 enzymes, the role of UGTs and SULTs in drug metabolism has received little attention. Liver microsomes, S9 fractions, and cryopreserved hepatocytes from human, dog, cynomolgus monkey, mouse, and rat were used as matrices in the study. Single compound, 7-hydroxycoumarin (7-HC), along with necessary cofactors was dosed into the matrices and incubated at 37° C; formation of two metabolites, 7-HC-glucuronide and 7-HC-sulfate, was determined with liquid chromatography with tandem mass spectrometry. Within the same species, the UGTs activities in microsomes and S9 fractions were comparable. In addition, UGTs activities in cryopreserved hepatocytes were lower than in the other matrices. Also, the SULTs activities were much higher in S9 fractions than in cryopreserved hepatocytes and microsomes. Species differences on UGTs and SULTs activities were also observed. The results indicated that S9 fractions, microsomes, and cryopreserved hepatocytes might be useful for UGTs metabolism study, whereas S9 fractions appear to be the most appropriate matrix for both UGTs and SULTs metabolism. Species differences with respect to phase II metabolism also need to be taken into consideration when selecting an in vitro system to evaluate various aspects of drug metabolism.