The present invention provides improved reverse hybrid assay methods for identifying amino acid residues within a protein that are required for its interaction or physical association with another protein, wherein disruption of an interaction between a protein of interest and its binding partner protein is assayed for a library of mutations of said protein of interest, and maintenance of an interaction between the protein of interest and another binding partner is assayed simultaneously in a single step, thereby reducing the incidence of uninformative mutations in the protein of interest that are detected.