Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are crucial redox cofactors regulating energy metabolism, reductive biosynthesis and the antioxidant defense system. Besides their role as redox cofactors, NAD(P)s have more recently also been described as important signaling molecules, acting for example as cosubstrate of different enzymes such as the sirtuins and poly(ADP-ribose) polymerases (PARPs), involved in the control of important cellular functions. Highlighting their important role, an imbalance of their intracellular concentration has been associated with aging and different pathologies. Therefore, the development of methods enabling the accurate subcellular quantification of NAD(P) in live cells is of great importance. This thesis describes the development and application of two FRET-based semisynthetic biosensors for NADPH/NADP+ and NAD+ in live cells. These sensors, named NAD(P)-Snifits, possess a high dynamic range, high specificity, are pH insensitive and can be excited at long wavelenghts. By using ratiometric imaging and fluorescence lifetime imaging, we quantified free NADPH/NADP+ and NAD+ in different subcellular compartments in live cells. We report here, for the first time, that free NADPH/NADP+ is significantly higher in the mitochondria than in the cytosol and observed moderately higher NAD+ level in the mitochondrial matrix consistent with previous report. We have also demonstrated that NADP-Snifit can be used to monitor the dynamics of NADPH/NADP+ caused by oxidative stress using conventional widefield fluorescence microscopy. The easy handing and use of these sensors make it possible to study not only the kinetics of cellular H2O2 scavenging and NADPH regeneration rate, but also to monitor drug-related side effects. Finally, we measured the effect of different conditions and drug treatment mimicking calorie restriction on free cytosolic and mitochondrial NADPH/NADP+ and NAD+ by flow cytometry. We observed that all these treatments increased free cytosolic and mitochondrial NAD+ concentrations with various levels of efficiency, but had divergent effects on the different subcellular NADPH/NADP+. NAD(P)-Snifits have the potential of becoming valuable tools to study the NAD(P) metabolism in physiological relevant conditions. Their wide applicability, in particular with high-throughput methods such as flow cytometry opens the possibilities for their use in metabolic and drug screening of large libraries.