The NAD/NADH-Glo Assay is a bioluminescent, homogeneous single-reagent-addition assay for detecting total oxidized and reduced nicotinamide adenine dinucleotides (NAD+ and NADH, respectively) and determining their ratio in biological samples or in defined enzyme reactions. An NAD Cycling Enzyme is used to convert NAD+ to NADH. In the presence of NADH, the provided reductase enzyme reduces a proluciferin reductase substrate to form luciferin. Luciferin then is quantified using Ultra-Glo Recombinant Luciferase, and the light signal produced is proportional to the amount of NAD+ and NADH in the sample. Cycling between NAD+ and NADH by the NAD Cycling Enzyme and Reductase increases assay sensitivity and provides selectivity for the nonphosphorylated NAD+ and NADH compared to the phosphorylated forms NADP+ and NADPH. The NAD Cycling Enzyme, Reductase and luciferase reactions are initiated by adding an equal volume of NAD/NADH-Glo Detection Reagent, which contains NAD Cycling Enzyme and Substrate, Reductase, Reductase Substrate and Ultra-Glo Recombinant Luciferase, to an NAD+- or NADH-containing sample. Detergent present in the reagent lyses cells, allowing detection of total cellular NAD+ and NADH in a multiwell format with addition of a single reagent. An accessory protocol is provided to allow separate measurements of NAD+ and NADH, and calculation of the NAD+ to NADH ratio. The simple add-mix-read protocol and scalable assay chemistry make the NAD/NADH-Glo Assay well suited to monitor effects of small molecule compounds on NAD and NADH levels in high-throughput formats.