Fluorescence polarization (FP) technology is based on the measurement of molecule rotation, and has been widely used to study molecular interactions in solution. This method can be used to measure binding and dissociation between two molecules if one of the binding molecules is relatively small and fluorescent. The fluorescently labeled small molecule (such as a small peptide) rotates rapidly in the solution. Upon excitation by polarized light, the emitted light remains depolarized and gives rise to a low FP signal. When the fluorescent small molecules in solution are bound to bigger molecules (such as a protein), the movement of the complex becomes slower. When such a complex is excited with polarized light, much of the emitted light is polarized because of the slow movement of the complex. Thus, the binding of a fluorescently labeled small molecule to a bigger molecule can be monitored by the change in polarization and measured by the generation of an increased FP signal. This chapter aims to provide a step-by-step practical procedure for developing an FP assay in a multi-well plate format to monitor protein-protein interaction (PPI) in a homogenous format.