Recent large-scale studies have provided a global description of the interactome-the whole network of protein interactions in a cell or an organism-for several model organisms. Defining protein interactions on a proteome-wide scale has led to a better understanding of the cellular functions of many proteins, especially those that have not been studied by classical molecular genetic approaches. Here we describe the resources, methods, and techniques necessary for generation of such a proteome-scale interactome in a high throughput manner. These procedures will also be applicable to low or medium throughput focused studies aimed at understanding interactions between members of specific pathways such as Notch signaling.