In one approach, sample is extracted and the extraction is aliquotted into 2 parts. One aliquot is treated at 65uC to degrade NAD+ and NVP-BKM120 subsequently measured for NADH only; meanwhile the other aliquot, which is not heat treated, can be assayed for the sum of NADH and NAD+. In the other approach, the same sample is divided and extracted in two different solutions: the alkali extraction for NADH and the acid extraction for NAD+. Both extractions will then be adjusted to neutral pH prior to performing the recycling assay to determine the concentration of pyridine nucleotides. In this study, we developed a method to extract total NADx from whole fruit flies while minimizing enzymatic degradation during sample preparation. We also modified the existing extraction procedure so that both oxidized and reduced state can be measured from the same homogenate and NAD+ /NADH ratio can be directly calculated, saving the effort of introducing an external control if NAD+ and NADH are extracted separately. We found this approach to be also suitable for assaying NADPH and NADP+ with small changes in the protocol. For the NADx assay that relies on ADH, we found a simple way to greatly improve the reaction linearity and assay sensitivity for this enzyme over a wide range. Direct measures of the quantity or concentration of pyridine nucleotides can be achieved in many ways but enzymatic recycling assays offer unique advantages. As NAD recycles between the redox indicator dye reaction. The signal is therefore vastly amplified and high sensitivity can be achieved. Furthermore, an enzymatic assay is highly versatile and specific. By changing the dehydrogenase reaction this assay can be adopted for detecting different redox coenzymes and in this report both NADx and NADPx assay are shown. By replacing PES/MTT with PMS/resazurin, it can be turned into a sensitive fluorescence assay as well. Because the reaction depends on dehydrogenase, specificity can be granted by carefully selecting dehydrogenase specific to only NAD+ or NADP+. In rats, Williamson et al. reported that during a shift from well-fed to starved, the free concentration ratio/changes as follows: from 725 to 528 in cytosol and from 8 to 5 in mitochondria. They also showed that the ratio of total NAD+ / NADH was 7.2 in cytoplasm and 2.2 in mitochondria hence most of NADH are in mitochondria and protein bound. The result regarding the total amounts of pyridine nucleotides was obtained using a method contributed by Glock and McLean, in which in order to measure total amount of pyridine nucleotides, sample was divided in two parts and extracted separately. One extraction is made in acid for assaying NAD+ and the other in alkaloid for NADH assay. Using the enzyme cycling method, our finding of total NAD+ /NADH being around 8 and halved with starvation agrees with these findings. NADPx predominantly exists in reduced form; we found the ratio of oxidized over reduced form to be around 0.2 for well-fed and decreased with starvation. The concentrations of PMS and resazurin in this study are carefully chosen based on the study of reaction mechanism by Candeias et al. It was shown in that report that when the concentration of PMS exceeds 1000 mM, it can form secondary products. Note that the low concentration of dye will certainly limit the upper detection range. This assay depends on the pH-dependent instability of pyridine nucleotides to distinguish between NAD+ from NADH.