Cell adhesion and migration are dynamic, complex processes that require tight temporal control of signalling cascades at defined subcellular sites to occur. Decades of research have used biochemical methods to identify numerous signalling pathways that are involved in the coordination of adhesion, membrane protrusion and contractility that all contribute to migration. However, understanding the way in which these signals are controlled within discrete sites in individual cells undergoing migration is essential to enable a clearer understanding of how these pathways are integrated during motility. Recent advances in the generation of fluorescent protein variants have enabled the development of probes to analyse localized changes in protein conformation, activation or interactions in single cells using microscopy. Many of these probes are based on the use of fluorescence resonance energy transfer that permits precise determination of interacting species based on the emission or lifetime properties of the fluorophores involved. Here, we provide an overview of some of the recent studies that have developed such probes and examples of how these biosensors have been used to further our understanding of cell migration signalling.