Protein and genetic interaction maps can reveal the overall physical and functional landscape of a biological system. To date, these interaction maps have typically been generated under a single condition, even though biological systems undergo differential change that is dependent on environment, tissue type, disease state, development or speciation. Several recent interaction mapping studies have demonstrated the power of differential analysis for elucidating fundamental biological responses, revealing that the architecture of an interactome can be massively re-wired during a cellular or adaptive response. Here, we review the technological developments and experimental designs that have enabled differential network mapping at very large scales and highlight biological insight that has been derived from this type of analysis. We argue that differential network mapping, which allows for the interrogation of previously unexplored interaction spaces, will become a standard mode of network analysis in the future, just as differential gene expression and protein phosphorylation studies are already pervasive in genomic and proteomic analysis.