Nicotinamide adenine dinucleotide (NAD⁺) is an essential substrate for sirtuins and poly(adenosine diphosphate–ribose) polymerases (PARPs), which are NAD⁺-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD⁺ concentrations within these subcellular compartments are thought to regulate the activity of NAD⁺-consuming enzymes; however, the challenge in measuring compartmentalized NAD⁺ in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD⁺ concentrations in subcellular compartments. We found that the concentrations of free NAD⁺ in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD⁺ biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD⁺ concentrations.