Microscale thermophoresis (MST) is a simple but powerful tool to study the in vitro interaction among biomolecules, and to quantify binding affinities. MST curves describe the change in the fluorescence level of a fluorescent target as a result of an IR-laser-induced temperature change. The degree and nature of the change in fluorescence signal depends on the size, charge, and solvation shell of the molecules, properties that change in function of the binding of a ligand to the fluorescent target.We used MST to describe the interaction between components of a regulatory module involved in plant cell wall integrity control. This module comprises the secreted peptide Rapid Alkalinization Factor 23 (RALF23) and its receptor complex consisting of the GPI-anchored receptor Lorelei-Like Glycoprotein 1 (LLG1) and a receptor kinase of the CrRLK1L family, FERONIA. Here we show how MST can also be used to study three-partner interactions.