Protein kinases are crucial components of intracellular signaling pathways which transmit signals by phosphorylation of downstream targets, altering their function. Efficient signal transduction requires precise kinase regulation within specific biological contexts, making tools that allow study of their dynamics in situ critical for understanding kinase function. Highlighted in this article is the design of genetically-encodable, FRET-based kinase biosensors with examples of their implementation to study kinase regulation in live biological contexts with high spatial and temporal resolution.