Protein post-translational modification (PTM) represents an important functional regulatory mechanism in the cell. Technological advances in the field of LC-MS/MS-based quantitative proteomics have enabled massively parallel detection and identification of novel PTMs in the past few decades. However, conventional biochemical approaches to characterize PTM function remain labourious and low throughput, and we lack general methods with which to predict, or even prioritize, which modification sites are likely to be functional in the proteome. Here we developed a quantitative proteomic method, termed “Hotspot Thermal Profiling” (HTP), to enable the unbiased, global, and high-throughput measurements of protein stability changes in response to site-specific phosphorylation events in live cells. Our proof-of-concept dataset showed that phosphorylation-induced thermal stability shifts can serve as a readout on the biophysical and functional effect of a phosphosite. HTP measured thermal stability profiles of thousands of bulk, unmodified proteoforms and phosphomodiforms, and revealed trends related to protein function and structure. HTP rediscovered well-known phosphorylation-mediated protein-protein interactions in protein 4EBP1 and enabled discovery of previously uncharacterized PTM-mediated interaction GAPDH and Vinculin. In vitro functional experiments of GAPDH and Vinculin validated and elucidated the molecular functions of Next, we developed two HTP method variants with aims to improve precision in sample processing, expand the depth of (phospho)proteome coverage, and increase the accuracy in LC-MS/MS quantitative measurements. We termed these method variants “Label-Enrich”- and “Label-Fractionate-Enrich”-HTP and demonstrated that the thermal stability of different proteoforms be reliably measured in these variant methods. Lastly, we explored the application of different thermal profiling strategies to profile protein-metabolite interactions in cells. We used a modified HTP workflow with an additional fractionation step and developed a SILAC-based thermal stability method to interrogate the proteome under acute-glucose withdrawal in a kinetic series. The resultant dataset revealed the time-dependent shifts in thermal stabilities of thousands of proteins, including in glycolytic enzymes known to interact with glucose metabolites. Overall, these new profiling approaches provided novel mechanistic insights into molecular interactions in the cell, and we envision that the extension HTP methods to diverse organisms, cell types, PTMs, and kinetic states can expand our understanding of the role of PTMs in regulating protein structure, function and signal transduction.