Most proteins do not function on their own but as part of large signaling complexes that are arranged in every living cell in response to specific environmental cues. Proteins interact with each other either constitutively or transiently and do so with different affinity. When identifying the role played by a protein inside a cell, it is essential to define its particular cohort of binding partners so that the researcher can predict what signaling pathways the protein is engaged in. Once identified and confirmed, the information might allow the interaction to be manipulated by pharmacological inhibitors to help fight disease. In this review, we discuss protein-protein interactions and how they are essential to propagate signals in signaling pathways. We examine some of the high-throughput screening methods and focus on the methods used to confirm specific protein-protein interactions including; affinity tagging, co-immunoprecipitation, peptide array technology and fluorescence microscopy.