MilliporeSigma SimpliconTM Technology can be used to introduce multiple genes into cells with transfection via a single, synthetic, polycistronic, self-replicating RNA vector. The transfected cell line can be placed under selection to maintain expression of the proteins of interest for a limited number of cell divisions, while leaving no genetic footprint in the modified cell line. Recently, this technology was successfully utilized to introduce four, integration-free, reprogramming factors into human iPSC fibroblasts1. For this work, we created three SimpliconTM vectors expressing Phase I, human, xenobiotic metabolizing enzymes. The first vector expressed just a single cytochrome P450 enzyme (CYP3A4), to determine optimal expression conditions and activity profile in HepG2 cells and HEK293 cell lines which stably expressed human liver transporters. To perform this optimization, testosterone was used a substrate to measure 6-beta-hydroxytestosterone produced, via mass spectrometry. Subsequently, we replaced the iPSC reprogramming factors with five cytochrome P450 enzymes (e.g. CYP3A4, CYP2C9, CYP2E1, CYP1A2 and CYP2D6) in two differently designed vectors and tested each separately in a 96-well plate assay using the same cell lines. Activity of each enzyme was quantified by mass spectrometry of the metabolites produced from the substrates testosterone, tolbutamide, chlorzoxazone, phenacetin and buproprion. Relative expression of each enzyme was determined by qPCR measurement. Unlike the HepG2 cell line, to achieve substrate uptake, HEK293 cells were transfected to stably express liver transporters (i.e. OATP1B1 or OATP1B3). While cytochrome P450 metabolism varied with the substrate used, both cell lines show a 10-fold increase of testosterone metabolism over background in a 96-well plate format, after two hours of substrate incubation. Cell lines transfected with a SimpliconTM vector, scaled, frozen and subsequently assayed (6 days, total), demonstrated a similar metabolism profile to those assayed 48hrs post transfection. Currently, most commercial high-throughput cell based assay systems used for chemical toxicity screening utilize cells which lack metabolic competence. Here, the MilliporeSigma SimpliconTM Technology is used to create a cell line which expresses and shows metabolic activity of five human xenobiotic metabolizing enzymes, and can be scaled to screen thousands of chemical compounds in a 96-well plate format.