Cytochrome P450 (CYP, P450) and uridine 5’-diphospho-glucuronosyltransferase (UGT) play crucial roles in drug metabolism phase I and II, respectively. Our previous studies suggest that there are functional interactions between P450 and UGT. We previously established a co-expression system featuring CYP3A4 and UGT2B7 using baculovirus-infected insect cells. Commercial microsomes are available that individually express CYP3A4/NADPH P450 reductase (CPR) or UGT2B7. It would be much easier if we could evaluate the functional interaction of CYP3A4 and UGT2B7 by simply mixing the microsomes. To address this issue, we presently compared our established co-expression system with a simple microsome mixing system. Co-expressed UGT2B7 suppressed CYP3A4 activity. On the contrary, adding UGT2B7 microsomes to CYP3A4/CPR microsomes significantly enhanced CYP3A4 activity. The enhancement was systematic and strongly dependent on UGT2B7 microsomes, and was abrogated by detergent treatment. The collective results suggested that the enhancement of CYP3A4 activity resulted from a non-physiological interaction between CYP3A4 and UGT2B7, which were both expressed on different membranes. The phenomenon was distinguishable and hardly ever reflected the physiological interaction. This pitfall can be avoided by not using simple mixing procedures. In selecting experimental materials and methods depending on the subject of the study, a co-expression system should be applied in the analysis of functional P450-UGT interaction.