There are many possible complications associated with the concomitant use of herbs and medications, but limited information is available on herb-herb or herb-drug interactions. Paederia foetida Linn. (family: Rubiaceae) is utilized in the Indian traditional medicine. It exhibits various pharmacological properties, such as antidiabetic, analgesic, anti-inflammatory, antimicrobial, hepatoprotective, anthelmintic, antiulcer and antioxidant activities. The purpose of the present work was to investigate the inhibitory potential of the P. foetida ethanolic extract and its bioactive constituent lupeol on hepatic phase I drug metabolizing enzymes. The high performance thin layer chromatography was performed for qualitative analysis of various extracts of P. foetida. The effects of P. foetida extract on rat liver microsomes (RLMs) and individual cytochrome P-450 (CYP) isozymes (CYP3A4 and CYP2D6) were investigated using CYP450-carbon monoxide complex assay and fluorescence microplate assay, respectively. The ethanolic extract and lupeol (both at a concentration of 100 μg/mL) showed 45±3.3 and 44±3.8% inhibition of rat liver microsomes, respectively, which were significantly less than that of known inhibitor ketoconazole (74±5.4% inhibition at 100 μg/mL). The 50% inhibitory concentrations (IC₅₀) of ethanolic extract on CYP3A4 and CYP2D6 were 78±2.3 and 82±3.1 μg/mL, respectively, whereas its major bioactive constituent lupeol has IC₅₀ values of 83±2.0 and 84±2.6 μg/mL for CYP3A4 and CYP2D6, respectively. The results were of lesser magnitude compared to known inhibitors, ketoconazole and quinidine, respectively. The current study revealed that P. foetida has less inhibitory potential in comparison to that of known inhibitors, ketoconazole and quinidine, on two major drug metabolizing isozymes, CYP3A4 and CYP2D6. Thus, the use of P. foetida as a complementary or alternative medicine may be safe in regard to herb-drug interactions.