Nowadays, especially through the advent of systems biology, and the ability to generate predictive models, it is even more important to transform biology from a qualitative to a quantitative science. Interactions between proteins not only need to be identified, but their equilibrium rate constants need to be determined as well. Here we describe a very elegant and simple system to obtain quantitative data for protein-protein interactions using cell-free protein biosynthesis in combination with Microscale Thermophoresis. We have been able to characterize the interaction of Calmodulin with Ca2+ as well as its ligand M13 straight in the in vitro synthesis reaction. Furthermore, we are demonstrating the generic applicability of this approach with another set of proteins, an Antibody fragment and Cyclophilin A. All this work has been done without tedious expression and prior purification of the interacting proteins.