Abstract Introduction: Gain-of-function mutations in the ErbB4 receptor tyrosine kinase have been found in a significant fraction of melanoma cell lines that are dependent on ErbB4 for proliferation. However, there is a scarcity of therapeutics for treating these ErbB4-dependent tumors. Our drug discovery approach is based on the observation that the Q43L mutant of the ErbB4 agonist Neuregulin 2beta (NRG2b) functions as a partial agonist/antagonist at ErbB4. NRG2b/Q43L stimulates ErbB4 tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and competitively antagonizes agonist stimulation of ErbB4-dependent cell proliferation. ErbB4 partial agonists that function as antagonists at ErbB4 may hold promise as targeted therapeutics for ErbB4-dependent melanomas. Experimental Procedures: An automated phospho-ErbB4 sandwich ELISA and automated proliferation assays were developed and deployed to identify small-molecule compounds that are likely to function as ErbB4 partial agonists/antagonists. Hit molecules were then further evaluated for ErbB4 specificity and antiproliferative mechanism(s) of action. To examine specificity for ErbB4, we tested whether the hit molecules inhibit ErbB4-dependent and -independent proliferation of the same cell line. ErbB4 specificity was also evaluated by determining whether the hit molecules exert effects on any of the other three members of the ErbB family of receptor tyrosine kinases: EGFR, ErbB2, and ErbB3. The mechanism(s) of action for antiproliferative effects were investigated by determining whether the hit molecules inhibited known effectors that couple ErbB4 to cellular proliferation, whether they degraded ErbB4, and whether the inhibitory effects induced by the molecules were reversible. Results: High-throughput screening (HTS) strategies have been utilized to identify 20 small-molecule compounds that stimulate ErbB4 tyrosine phosphorylation, fail to stimulate ErbB4-dependent cellular proliferation, and inhibit agonist-induced ErbB4-dependent cellular proliferation. Efforts to determine whether these hits are specific for ErbB4 and by what mechanism they induce antiproliferative effects are under way and will be reported. Structures of these small-molecule ErbB4 partial agonists/antagonists may be reported, pending submission of a provisional patent application. Conclusions: Using validated HTS methodologies for identifying ErbB4 partial agonists that function as ErbB4 antagonists has led to the identification of 20 hits. Molecules that are specific for ErbB4 hold promise as targeted therapeutics for melanoma and other ErbB4-dependent tumors. Citation Format: Richard L. Cullum, Lauren M. Lucas, John T. Piazza, Jared I. Senfeld, Logan T. Neel, Ram B. Gupta, Allan E. David, David J. Riese. Characterization of putative ErbB4 antagonists: targeted melanoma drug discovery [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A159.