The yeast two-hybrid assay is a system for identifying and analysing protein-protein interactions. Since the original description in 1989, the technique has provided insight into many biological pathways. A variety of adaptations to the technique have been developed that allow analysis of protein-DNA, protein-RNA, or small molecule-protein interactions. Recent developments now allow the use of these technologies to perform global analyses of all such interactions that occur in cells. The information gained from these approaches is uncovering many aspects of the complex networks that underlie normal cellular processes and how they are perturbed in disease states.