The advent of induced pluripotent stem cell (iPSC) technology has enabled the modeling of an array of specific human disease phenotypes, aiding in the increasingly important and indispensable understanding of disease progression and pathogenesis. Pluripotent stem cell-derived hepatocytes present a new avenue for drug screening and personalized drug testing toward precision medicine. CYP450 microsomal enzymes play a critical role in drug metabolism. Hence, CYP activity measurement of iPSC-derived hepatocytes is a vital prerequisite, to ensure metabolic functionality before proceeding to drug testing. Herein, we describe the protocol for measurement of different CYP450 enzyme activities in human iPSC-derived hepatocytes.