Surface plasmon resonance (SPR) is one of the most commonly used techniques to study protein–protein interactions. The main advantage of SPR is it gives on the ability to measure the binding affinities and association/dissociation kinetics of complexes in real time, in a label-free environment, and using relatively small quantities of materials. The method is based on the immobilization of one of the binding partners, called the ligand, on a dedicated sensor surface. Immobilization is followed by the injection of the other partner, called the analyte, over the surface containing the ligand. The binding is monitored by subsequent changes in the refractive index of the medium close to the sensor surface upon injection of the analyte. During the last 10 years, SPR has been intensively used in the study of secretion systems because of its ability to detect highly dynamic complexes that are difficult to investigate using other techniques. This chapter will guide users in the setup of SPR experiments in order to identify protein complexes and to assess their binding affinity or kinetics. It will include detailed protocols for (i) the immobilization of proteins with the amine coupling capture method, (ii) analyte-binding analysis, (iii) affinity/kinetic measurements, and (iv) data analysis.