Proteins are central drivers of physiological and pathological processes in the cell. Methods evaluating protein functional states are therefore vital to fundamental research as well as drug discovery. Thermal proteome profiling (TPP) to this date constitutes the only approach that permits examining protein states in live cells, under native conditions and at a proteome-wide scale. TPP harnesses ligand/perturbation-induced changes in protein thermal stability, which are monitored by multiplexed quantitative mass spectrometry. In this chapter, we describe a modular experimental workflow for TPP experiments using live cells or crude cell extracts. We provide the tools to perform different TPP formats, i.e., temperature range experiments, TPP-TR; isothermal compound titrations, TPP-CCR; and a combination thereof, 2D-TPP.