The NAD/NADH-Glo and NADP/NADPH-Glo Assays are bioluminescent, homogeneous, single-reagent-addition methods to rapidly detect NAD+ and NADH and NADP+ and NADPH levels in cells and enzymatic reactions. Current methods to detect nicotinamide adenine dinucleotides lack sensitivity and reproducibility and require sample manipulation and large numbers of cells for cell-based applications. The assay is easily adapted for inhibitor screening in high-throughput formats. The sensitivity and robustness of the assay chemistry allow fewer cells to be used to detect individual nucleotides directly in multiwell plates or analysis of enzymes with low activity or with low Km values.