ITC is based on binding-mediated temperature changes and thus is unique among biophysical methods because it allows direct insights into the thermodynamics of the binding event. In a single experiment, thermodynamic parameters, such as enthalpy (ΔH), entropy (ΔS), and free energy (ΔG), are determined in addition to the steady-state affinity (KD) and stoichiometry. Furthermore, ITC is one of the few biophysical techniques that allows for the investigation of molecules in solution and without the need for any modification of the investigated molecules. The technology is compatible with a broad range of different buffer compositions, increasing its applicability in a variety of settings. With many years of experience, we apply ITC to thermodynamically profile hit clusters and stereoisomers, support hit-to-lead projects, and nominate hits for structural biology. Beyond protein–ligand interactions, we use ITC to characterize RNA–ligand binding.