The numerous biological roles of NAD⁺ are organized and coordinated via its compartmentalization within cells. The spatial and temporal partitioning of this intermediary metabolite is intrinsic to understanding the impact of NAD⁺ on cellular signaling and metabolism. We review evidence supporting the compartmentalization of steady-state NAD⁺ levels in cells, as well as how the modulation of NAD⁺ synthesis dynamically regulates signaling by controlling subcellular NAD⁺ concentrations. We further discuss potential benefits to the cell of compartmentalizing NAD⁺, and methods for measuring subcellular NAD⁺ levels.