Here, we describe an optimized protocol to identify specific nuclear receptor ligands. First, to rule out any compound interference with luciferase activity per se, we describe an in vitro assay assessing potential inhibition or activation of luciferase enzymatic activity. Second, to comply with EMA and FDA guidelines to mitigate drug-drug interactions, we detail assays assessing CAR and PXR activation ability. Finally, to minimize off-target detection effects, we describe the use of mammalian one-(or two-) hybrid systems. For complete details on the use and execution of this protocol, please refer to Hering et al. (2018).