Abstract Thermal proteome profiling (TPP) is based on the principle that, when subjected to heat, proteins denature and become insoluble. Proteins can change their thermal stability upon interactions with small molecules (such as drugs or metabolites), nucleic acids or other proteins, or upon post‐translational modifications. TPP uses multiplexed quantitative mass spectrometry‐based proteomics to monitor the melting profile of thousands of expressed proteins. Importantly, this approach can be performed in vitro , in situ , or in vivo . It has been successfully applied to identify targets and off‐targets of drugs, or to study protein–metabolite and protein–protein interactions. Therefore, TPP provides a unique insight into protein state and interactions in their native context and at a proteome‐wide level, allowing to study basic biological processes and their underlying mechanisms.