Mass spectrometry has revolutionized the ability to study posttranslationally modified proteoforms from biologic samples, yet we still lack methods to systematically predict, or even prioritize, which modification sites may perturb protein function. This talk will describe a novel proteomic method to detect the effects of site-specific protein phosphorylation on the thermal stability of thousands of native proteins in live cells. This massively parallel biophysical assay revealed surprising shifts in overall protein stability in response to site-specific phosphorylation sites, as well as trends related to protein function and structure. This method, hotspot thermal profiling (HTP), detects both intrinsic changes to protein structure as well as extrinsic changes to protein-protein, and protein-metabolite interactions resulting from the diminutive introduction of a phosphate onto large proteins. Finally, I will discuss how functional “hotspot” protein modification sites can be discovered and prioritized for study in a high-throughput and unbiased fashion, and characterized kinetically in response to k cellular signaling events. This approach should be applicable to diverse organisms, cell types and posttranslational modifications.