Background: Xenonbiotic and drug-metabolizing enzymes (DME’s) are involved in the bioconversion of xenobiotics including drugs, synthetic chemicals and environmental pollutants into inactive or active compounds. In pharmacological therapy, bioconversion can either lead to detoxification or activation of the drug, which has implications on treatment effectiveness and toxicity. Quantitative profiling of the drug-metabolizing sub-proteome can be used in the characterization of liver drug metabolism profiles in individual patients which can be a major step towards stratified or personalized medicine. Methods: Label-free quantification of approximately 70 drug-metabolizing enzymes in three individual human livers was carried out using a nanoLC-IMS-QToF-MS/MS method with an LC programme of 70 minutes. A range of cytochrome P450 and uridine 5'-diphosphate glucuronosyltransferase (which are the main drug metabolizing enzymes in the liver) were quantified previously using QconCAT SRM assays  to externally validate the label-free quantification.Results: The label free approach showed a high level of precision and reproducibility. The concentrations of the liver enzymes quantified using the two methods (untargeted vs. targeted proteomics) were shown to agree (R2 = 0.50, Rs = 0.80, p <0.0001***, n = 38 enzymes, three individual livers). A range of 70 drug-metabolizing enzymes were quantified.Conclusion: The label free methodology described here can be used for the global profiling of enzymes and transporters in different biological in vitro and in vivo systems relevant to pharmacology.  Achour B, Russell MR, Barber J, Rostami-Hodjegan A (2014) Simultaneous quantification of the abundance of several cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes in human liver microsomes using multiplexed targeted proteomics. Drug Metab Dispos 42(4): 500-510.