<p><i>Haemonchus contortus</i> is a pathogenic nematode that infects small ruminants. Chemotherapy is the main treatment for these parasitic infections, but the rapid rise of drug resistance calls for the development of new anthelmintics. To support this, optimizing screening assays is vital for identifying new drugs. The exsheathed L3 (xL3) stage of <i>H. contortus</i> is often used in in vitro evaluations; however, it has been observed that it is less sensitive than the adult stage, possibly due to enhanced detoxification pathways. To explore this hypothesis, inhibitors of xenobiotic detoxification pathways were tested on the activity (IC₅₀) of four anthelmintics—monepantel (MOP), levamisole (LEV), ivermectin (IVM), and albendazole sulfoxide (ABZ SO)—in xL3 using an automated motility assay. The inhibitors used were piperonyl butoxide (PBO) for phase I metabolism, 5-nitrouracil (5-NU) for phase II metabolism, and zosuquidar (ZOS) inhibiting efflux transport proteins. PBO increased MOP IC₅₀, likely due to reduced formation of the active metabolite monepantel sulfone. IC₅₀ of MOP with 5-NU and IVM with PBO were both diminished, suggesting differences in metabolism between xL3 and the existing reports for the adult stage. Coincubation of LEV and IVM with ZOS also reduced IC₅₀, confirming previous studies. ABZ SO was unaffected by the inhibitors. The use of inhibitors of xenobiotic detoxification pathways led to significant changes in the in vitro activity of the anthelmintics evaluated in <i>H. contortus</i> xL3 stage. Further studies, as ex vivo parasite diffusion assays in the xL3 stage, should be conducted to directly assess the impact on detoxification pathways.</p> Graphical Abstract <p></p>