Protein kinases are central players in almost every aspect of human physiology and serve as critical mediators of signal transduction. The dysfunction of many kinases is implicated in cancer, diabetes, inflammation, and cardiovascular diseases and many are validated targets for therapeutic intervention. The human genome encodes ~518 protein kinases. Yet, the 25 or so marketed protein kinase drugs, target only a small fraction of the human kinome. A greater portion remains unexplored, with very little information available on their role in cell signaling. Chemical tools can play a pivotal role in target discovery and validation. Selective and potent small-molecule kinase inhibitors can be used to address this knowledge gap by pharmacological knockdown of a target protein-kinase in their native environment and probe the effect of inhibition on cell signaling and disease pathology. Our goal is to systematically focus on the vast number of historically understudied kinases, which are currently non-druggable due to lack of mechanistic and functional understanding due to a paucity of reagents and associated assays to gauge selectivity, This gap will be addressed by high-quality chemical probe development, capable of selectively interrogating function of target kinases. During the Phase II period, we will iteratively a) develop low-cost, robust assays using our platform split-luciferase technology for the understudied kinases and, b) develop high- quality chemical probes, which can help deconvolute kinase biology and target discovery. The chemical probe sets developed under this grant will be available as a resource to the research community.