AbstractGenerating reference maps of interactome networks illuminates genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. We apply state-of-the-art methods to identify binary protein-protein interactions (PPIs) forDrosophila melanogaster. Four all-by-all yeast two-hybrid (Y2H) screens of > 10,000Drosophilaproteins result in the ‘FlyBi’ dataset of 8723 PPIs among 2939 proteins. Testing subsets of data from FlyBi and previous PPI studies using an orthogonal assay allows for normalization of data quality; subsequent integration of FlyBi and previous data results in an expanded binaryDrosophilareference interaction network, DroRI, comprising 17,232 interactions among 6511 proteins. We use FlyBi data to generate an autophagy network, then validate in vivo using autophagy-related assays. Thedeformed wings(dwg) gene encodes a protein that is both a regulator and a target of autophagy. Altogether, these resources provide a foundation for building new hypotheses regarding protein networks and function.