In this article we describe the validation of a beta-lactamase reporter assay for high-throughput interrogation of the mitogen-activated protein kinase (MAPK) signaling pathway initiated by multiple receptors. Activation of cell surface receptors, such as epidermal growth factor receptor (EGFR) and cMET, upon ligand binding, leads to the activation of the downstream MAPK signaling pathway and transcription factor, activator protein 1 (AP1), which then induce the expression of genes that are important for cell growth and proliferation. Our MAPK pathway reporter cell line, AP1-bla ME-180, expresses multiple endogenous cell surface receptors. We demonstrate that this reporter assay can be used to monitor the MAPK pathway initiated by cell surface receptors, including EGFR, cMET, and tumor necrosis factor receptor. Our results from Stealth (Invitrogen Corp., Carlsbad, CA) RNA interference and small molecule inhibitor studies suggest that activation of individual kinases of the MAPK pathway is regulated in a ligand-specific manner. The AP1-bla ME-180 reporter cell line can therefore be used to screen for compounds with desired selectivity profiles.