Microscale thermophoresis (MST) is a technology for the biophysical analysis of interactions between biomolecules. Microscale thermophoresis is based on the detection of a temperature-induced change in fluorescence of a target as a function of the concentration of a non-fluorescent ligand. The observed change in fluorescence is based on two distinct effects. On the one hand it is based on a temperature related intensity change (TRIC) of the fluorescent probe, which can be affected by binding events. On the other hand, it is based on thermophoresis, the directed movement of particles in a microscopic temperature gradient. Any change of the chemical microenvironment of the fluorescent probe, as well as changes in the hydration shell of biomolecules result in a relative change of the fluorescence detected when a temperature gradient is applied and can be used to determine binding affinities. MST allows measurement of interactions directly in solution without the need of immobilization to a surface (immobilization-free technology).