Abstract Chronic pain is one of the leading health problems worldwide. Opioids have served as gold standard for treating moderate to severe pain. However, opioids possess serious adverse effects that limit their use in clinics. Therefore there is an unmet need for alternative effective and non-addictive pain treatments. Neuropeptide B/W Receptor 1 (NPBWR1) has emerged as a novel pain target which upon activation can produced analgesic effects on its own or in synergy with opioids. To date, all reported NPBWR1 agonists are peptides or peptidomimetics that do not cross the blood brain barrier, requiring central administration. To facilitate research on this promising target, we propose to perform a high throughput screen of our diverse small molecule library to identify small molecule agonists (Aim 1). We have developed a battery of in vitro functional assays to characterize NPBWR1 ligands including a cAMP, calcium mobilization and TruPath assays. The 384-well cAMP assay has been demonstrated to have good robustness Z'>0.5, S/B > 20. Hits will be validated using counter screen and orthogonal assays. Validated hits will be assessed for ADME profiling and selectivity against other targets (Aim 2). Completion of this proposal will yield small molecule NPBWR1 that serve as starting points for subsequent medicinal chemistry optimization to develop into therapeutics for chronic pain and other NPBWR1-mediated conditions.