Organisms must maintain the integrity of their genomes. Cells continuously sustain DNA damage from both endogenous and exogenous sources, and unaddressed or improperly repaired or removed lesions can lead to mutations, oncogenesis, or cell death. The size and complexity of the genome combined with the innumerable sources of genotoxicity make timely DNA repair a formidable task. Over millions of years, Eukaryotic evolution has produced intricate signaling networks that coordinate cellular machinery to both prevent and correct DNA damage. Central to this coordinated response are the phosphatidylinositol 3-kinase-related kinases (PIKKs): ATR/Mec1, ATM/Tel1, and DNA-PKcs. Each is preferentially activated by distinct forms of DNA damage, initiating signaling cascades that orchestrate outcomes ranging from the repair of a single DNA break to the arrest of the entire cell cycle in service of maintaining genomic stability.To enact these sweeping changes in cellular behavior, kinases apply hundreds of phosphorylation events on diverse substrates, including DNA repair proteins, transcription factors, and metabolic regulators. Characterizing these signaling networks is analytically challenging due to their immense complexity and the low i stoichiometry of many phosphorylation events. Mass spectrometry has emerged as an essential tool in this space, enabling unbiased identification and quantification of thousands of phosphopeptides in a single experiment. Chapter 1 will review the trajectory of mass spectrometry-based phosphoproteomics as a robust tool for studying DNA damage signaling, and Chapter 2 will describe my efforts to optimize and implement a phosphoproteomic pipeline for profiling the signaling network of Tel1, the yeast homologue of the PIKK ATM. While phosphoproteomic screens can reveal hundreds of phosphorylation events, they often lack spatial context. The cell is not a uniform space, and the spatial distribution of phosphorylation events is crucial to the biological outcome of signaling responses. Although fluorescent biosensors can provide spatially resolved readouts, they typically monitor only a single phosphorylation site, limiting their utility for broader network-level insights. Chapter 3 will detail my efforts to develop and implement a technique for quantitatively monitoring the activity of multiple kinases simultaneously with spatial resolution. This technique has been dubbed ProKAS: proteomic kinase activity sensors. Through the use of a protein biosensor containing localization signal peptides (“targeting elements”) and kinase-specific phosphorylation targets, ProKAS allows us to use mass spectrometry to quantify the activity of kinases of interest at different compartments or subcellular locations. Together, these studies describe how I built upon existing phosphoproteomic workflows to achieve deeper coverage of kinase signaling networks and developed a novel platform for multiplexed, spatially resolved monitoring of kinase activity.