Protein-protein interactions are a preliminary but fundamental key to many biological systems. Identification of proteins that interact with particular bait not only contributes to a deeper understanding of bait protein function but also provides much information for the discovery of larger-scale interaction networks (interactome). Therefore, protein-protein interaction mapping is regarded as a widely accepted standardized functional genomics technique that provides comprehensive functional interpretation of previously uncharacterized proteins. A commonly used approach to detecting novel protein-protein interactions is the yeast two-hybrid system. In this chapter we describe in detail the protocols used to dissect the rice MAPK interactome, including the bait protein auto-activation test, identification of a rice MAPK interacting protein, confirmation of interaction by retransformation assay and characterization of the novel interacting protein.