It is important to understand the potential of botanical-drug interactions to ensure the safe use of botanical dietary supplements (BDS). Cytochrome P450 (P450) is one of the most abundant phase 1 drug-metabolizing enzymes and is accountable for a great deal of pharmacokinetic botanical-drug interactions. This problem is particularly acute for older adults who often consume BDS with multiple prescription medicines. The consequences of botanical-drug interactions can lead to lack of prodrug efficacy or drug toxicity from reduced drug clearance through inhibition of P450 metabolizing enzymes. In this study, a 7-in-1 cocktail P450 inhibition assay with 7 Food and Drug Administration-recommended P450s (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4/5) including CYP2B6 recombinant enzyme was performed, minimizing substrate interactions with respect to specificity while maximizing assay sensitivity. High-performance liquid chromatography-mass spectrometry was used for quantitative determination of probe substrate metabolism. Withania somnifera L. Dunal (ashwagandha), a popular BDS in the United States with sales of ∼ 6 million in 2021, is used to promote sleep and relieve stress and anxiety, especially in older adults. However, comprehensive studies of pharmacokinetic drug interactions with ashwagandha, especially with leaf extracts, have not been reported. Four extracts from ashwagandha root or leaf were evaluated for P450 inhibition, and no reversible inhibition was detected at IC