The US EPA’s ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to false positive (chemical is detoxified in vivo) as well as false negative results (chemical is bioactivated in vivo) and thus potential mischaracterization of chemical hazard. We have addressed this challenge by introducing the ten most prevalent human liver cytochrome P450 (CYP) enzymes into a human cell line (HEK293T) with low endogenous metabolic capacity. The CYP enzymes were introduced via transfection of modified mRNAs as singlets or as a mixture in relative proportions expressed in the liver. Initial experiments using luminogenic CYP450 substrates demonstrate that cell models express metabolic enzymes from the transfected mRNAs and activities are significantly increased when co-transfected with a CYP accessory protein, P450 oxidoreductase (POR). Transfected HEK293T cells demonstrate the ability to produce predicted metabolites following treatment with well-studied CYP substrates, with metabolite formation occurring through 18 hours post-treatment. As a demonstration of how this method can be used to retrofit existing HTS assays, a proof-of-concept screen for cytotoxicity in HEK293T cells was conducted using 56 test compounds. The results demonstrate that the xenobiotic metabolism conferred by transfection of CYP-encoding mRNAs shifts the dose-response relationship for certain test chemicals such as aflatoxin B1 (bioactivation) and fenazaquin (detoxification). Overall, transfection of CYP-encoding mRNAs is an effective and portable solution for retrofitting metabolic competence to existing cell-based HTS assays. This dataset is associated with the following publication: DeGroot, D., A. Swank, R. Thomas, M. Strynar, M. Lee, P. Carmichael, and S. Simmons. mRNA transfection retrofits cell-based assays with xenobiotic metabolism. JOURNAL OF PHARMACOLOGICAL & TOXICOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, USA, 92: 77-94, (2018).