Although nicotinamide adenine dinucleotide phosphate (NADPH) is produced and consumed in both the cytosol and mitochondria, the relationship between NADPH fluxes in each compartment has been difficult to assess due to technological limitations. Here we introduce an approach to resolve cytosolic and mitochondrial NADPH fluxes that relies on tracing deuterium from glucose to metabolites of proline biosynthesis localized to either the cytosol or mitochondria. We introduced NADPH challenges in either the cytosol or mitochondria of cells by using isocitrate dehydrogenase mutations, administering chemotherapeutics or with genetically encoded NADPH oxidase. We found that cytosolic challenges influenced NADPH fluxes in the cytosol but not NADPH fluxes in mitochondria, and vice versa. This work highlights the value of using proline labeling as a reporter system to study compartmentalized metabolism and reveals that NADPH homeostasis in the cytosolic and mitochondrial locations of a cell are independently regulated, with no evidence for NADPH shuttle activity.