Genetically encoded fluorescent sensors specific for energy metabolites are starting to become key tools for studying brain energy metabolism at the single cell level. Sensors for glucose, glutamate, ATP, NADH, lactate, and pyruvate have been successfully used in brain cells and brain slices. Providing high spatiotemporal resolution, these sensors report cell-specific metabolite levels, absolute concentrations, and even metabolic fluxes, and are being instrumental for the discovery of new phenomena and their molecular underpinnings. Here, we report the methodology and first results from in vivo applications of sensors for glucose, lactate and pyruvate. Furthermore, we provide example applications for investigating cell-specific metabolic processes in the cerebral cortex of mice.