Coenzyme II is indispensable for biosynthetic reactions and antioxidant functions; nevertheless, ascertaining its role in the metabolism of living cells remains technically problematic. For more than 50 years, NADPH’s weak intrinsic fluorescence has been utilized to study the metabolic state of living tissues. Despite substantial knowledge of specific enzymes and pathways that employ these coenzymes, there is still much to learn about how NADH and NADPH-dependent pathways are regulated and compartmentalized. One of the challenges in investigating these processes in living cells has been the lack of suitable detection methods. This chapter aims to address this issue by providing information on both traditional methods and newly developed genetically encoded indicators for measuring NADP+ and NADPH.