The Ah receptor (AhR) is a ligand-dependent transcription factor that mediates a wide range of biological and toxicological effects from exposure to structurally diverse synthetic and naturally occurring chemicals. The role of the AhR and its signaling pathway in endogenous physiological functions and its involvement in immune cell development and human diseases has made it a target for development of therapeutic agents. The ability of the AhR to stimulate gene expression in a ligand-specific manner in recombinant mammalian cell lines containing a stably transfected AhR-responsive firefly luciferase or enhanced green fluorescent protein (EGFP) reporter gene permits high throughput chemical screening for AhR activators. The induction of luciferase activity or EGFP fluorescence in these readily available recombinant cell lines occurs in a time-, dose- and AhR-dependent and chemical-specific manner where the magnitude of reporter gene induction is directly proportional to the concentration and potency of the inducing chemical. The AhR agonist activity of positive test chemicals can be confirmed by demonstrating their ability to stimulate expression of CYP1A1, an endogenous AhR-responsive gene, using quantitative real-time PCR. The detailed protocols described here provide step-by-step instructions for detection and characterization of activators of AhR-dependent gene expression that can readily be applied to other appropriate cell lines.