The Src/FAK complex is involved in many signaling pathways and plays crucial roles in cell adhesion/migration. It becomes clear that the subcellular localization of Src and FAK is crucial for their activities and functions. In this article, we first overview the molecular mechanisms and functions of Src and FAK involved in cell adhesion/migration. We then introduce the development of genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) to visualize the activities of Src and FAK in live cells with high spatiotemporal resolutions. Different kinds of signal peptides targeting subcellular compartments are also discussed. FRET-based biosensors fused with these targeting signals peptides are further introduced to provide an overview on how these targeting signals can facilitate the localization of biosensors to continuously monitor the local activity of Src and FAK at subcellular compartments. In summary, genetically-encoded FRET biosensors integrated with subcellular compartment-targeting signals can provide powerful tools for the visualization of subcellular Src and FAK activities in live cells and advance our in-depth understanding of Src/FAK functions at different subcellular compartments.