A diverse series of mammalian two-hybrid technologies for the detection of protein-protein interactions have emerged in the past few years, complementing the established yeast two-hybrid approach. Given the mammalian background in which they operate, these assays open new avenues to study the dynamics of mammalian protein interaction networks, i.e. the temporal, spatial and functional modulation of protein-protein associations. In addition, novel assay formats are available that enable high-throughput mammalian two-hybrid applications, facilitating their use in large-scale interactome mapping projects. Finally, as they can be applied in drug discovery and development programs, these techniques also offer exciting new opportunities for biomedical research.