Budlein A (BUD_A) is a sesquiterpene lactone that was first isolated from the leaves of Viguiera buddlejiformis Benth. & Hook.f. ex Hemsl. It shows potent in vitro and in vivo anti-inflammatory effect as well as cytotoxic activity against tumor cell lines (Arakawa et al, 2013). Cytochrome P450 (CYP450) enzymes are a superfamily of heme containing mono-oxygenases that contribute to phase I metabolism of marketed drugs (Nirogi et al, 2015). In the drug selection process, it is critical to identify the CYP responsible for the metabolism in order to predict any potential drug interaction (Lu, Wang, Lin, 2003). The aim of the present study was to determine the recombinant CYP (rCYP) enzymes responsible for the BUD_A in vitro metabolism. The analysis of the samples was performed by LC-MS/MS. The incubation was performed in eppendorf tubes containing 200 μL microsomal medium composed of 3 μL BUD_A solution, 40 μL of each rCYP and 107 μL sodium phosphate buffer solution (pH 7.4; 0.1 mol L-1). The evaluated isoforms were: 1A2, 3A4, 3A5, 2B6, 2C8, 2C9, 2C19, 2D6 and 2E1 (50 pmol mL-1). The incubation medium was preheated (5 min, 37 °C) and the reaction was started after addition of 50 μL NADPH regeneration system solution. The in vitro metabolism was quenched by the addition of 1 mL ethyl acetate, 90 minutes later. Negative controls, without the isoforms, were prepared. All assays were performed in quintuplicate. For each rCYP, the metabolite formation rate was multiplied by the mean specific content of the corresponding CYP in native human liver microsomes, this yields the normalized rate (NR). The NR values for each rCYP were summed to obtain the total normalized rate (TNR). Finally, the NR for each rCYP was expressed as a percentage of the TNR (Rodrigues, 1999). The obtained results were analyzed using GraphPad Prism 5.0 software (San Diego, CA, USA). The correlation was considered statistically significant only when % TNR was greater than 30% (Rodrigues, 1999). We identified three oxidative metabolites in liver microsomes, namely M1 ([M+H]+ at m/z 293), M2 and M3 ([M+H]+ at m/z 391). The results suggest that the metabolism of BUD_A was catalyzed predominantly by the rCYPs 3A4 (M1, 39%; M2, 97%; M3, 99%) and 2C9 (M1, 61%). Data from CYP phenotyping experiments demonstrated that virtually all major CYPs are able to metabolize BUD, but that CYP3A4 is the main one.