Abstract: Ex vivo methods of detecting and analyzing circulating drug metabolites with drug interaction potential are provided. The methods include the use of clinical plasma samples from subjects who have been administered an investigational drug in vivo. The clinical plasma samples will contain both the parent drug and associated metabolites. A control plasma sample spiked directly with the drug of interest can be used as a standard reference. The plasma samples can be applied to an in vitro test system to evaluate the changes in activity or expression of drug-metabolizing enzymes and/or drug transporters in the test systems to determine circulating drug metabolites with drug interaction potential. By comparing the clinical plasma sample to the drug-spiked control, the inhibitory and/or inducing effects on drug-metabolizing enzymes and/or drug transporters can be correctly attributed to the parent drug or its associated metabolites.