Kinases are a central hub for signaling in multiple disease settings, including cancer, autoimmune disease, heart disease, and beyond. There is significant need for technologies that could streamline multiplexed measurement of kinase activities in live cells using high throughput screening and clinical lab-compatible read-outs for future translation. We develop these kinds of cell-based assay approaches, designing novel substrates tuned to particular read-out technologies and characterize them in vitro and in cell-based assays. Through prior work, we prototyped an in silico pipeline called KINATEST-ID, in which kinase substrate preferences are identified, cross- checked against other “off-target” kinases, then novel peptides are designed based on predicted compatibility with a read-out technique and tested empirically. Our initial iterations of this pipeline have focused on tyrosine kinases and in vitro lanthanide fluorescence assay read-outs, which have limited multiplexability. Mass spectrometry detection of cell-deliverable kinase substrates that report kinase activity in live cells would provide far higher multiplexability, but in prior work we ran up against a fundamental physiochemical limitation of our design pipeline: selecting sequences for Tb3+ chelation produces substrates biased towards acidic amino acids (e.g. D, E) that ionize very poorly in standard MS analyses. We also found that while we could predict biochemical efficiency for kinases that had high-quality preference data available, prediction of selectivity is still inadequate because most kinases lack such data for cross-referencing against each other. These barriers have limited further progress on developing substrates for cell-based kinase profiling assays, particularly with multiplexed MS detection. Further, in order to be more robust for higher throughput analyses, the workflows for the assay, sample processing, and MS detection need to be simplified. In Aim 1, we will expand the KINATEST-ID platform functionality with a focus on MS detection. In Aim 2, we will develop multiplexed cell-based deliverable substrate kinase assays using targeted parallel reaction monitoring (PRM) MS methods in collaboration with colleagues at Cedars Sinai who are developing cutting edge, high throughput proteomics methods that are clinical lab- compatible. This work will produce an optimized platform for developing and implementing MS-compatible, cell-based assays enabling kinase activity profiling in live cells, as well as a path to new tools for understudied kinases. Overall, these tools will have high potential to impact both basic research for rapid profiling of signaling activities, and kinase inhibitor drug discovery with eventual translation to the clinic.