In vivo cocktail pathway phenotyping (ICPP) is routinely used to assess the metabolic drug-drug interaction (mDDI) potential of new drug candidates (NDC) during drug development. However, there are a number of potential limitations to this approach and the use of validated drug cocktails and study protocols is essential. Typically ICPP mDDI studies assess only the impact of interactions following multiple postulated perpetrator doses and hence the emphasis in terms of validation of these studies has been ensuring that there are no interactions between probe substrates. Studies assessing the comparative impact of single and multiple doses of the postulated perpetrator have the potential to provide richer information regarding both the clinical impact and mechanism of mDDIs. Using modafinil as a model compound, we sought to develop an optimized ICPP mDDI study protocol to evaluate the potential magnitude and clinical relevance of mDDIs using a physiologically based pharmacokinetic modeling approach.