The purpose of this study was to characterize a novel optogenetic kinase system, a Light-Regulated (LightR) switch domain, designed to improve spatiotemporal control of signaling. Using LC-MS/MS, tyrosine phosphorylation was measured after 10 seconds, 30 seconds, 1 minute, 5 minutes and 60 minutes of LightR Src activation. TMT labeling was utilized to allow multiplexed relative quantification across time points. Results provide insight into the timing of Src and Src substrate activation.