Recent years have seen a surge of interest in the role of NAD in human disease and whether increasing NAD with supplements such as nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN) has therapeutic value. However, our understanding of NAD and its metabolites in vivo is limited by technical barriers, a lack of consensus on analytical techniques, and incomplete knowledge of the catabolic routes taken when NAD breaks down under various stressed states. For many studies, blood and urine are the only samples available to investigative teams. Whole blood contains NAD(P)(H), but poses important stability considerations for handling and measurements. Serum and urine are relatively stable, but contain mostly catabolites of NAD, which themselves carry implications for NAD metabolism in the rest of the body. Here we propose to take advantage of a multidisciplinary team to develop and standardize methods for the analysis of NAD, as well as NADH, NADP, NADPH that are suitable for human clinical trials. These include extensive validation and testing of methods for determining the abundance of NAD(P)(H) in blood-derived samples and comparisons between liquid chromatography-mass spectrometry-based assays, biochemical assays, and nuclear magnetic resonance. We will also perform the first direct comparison of the two major magnetic resonance spectroscopy (MRS) methods that have been used to detect NAD in intact tissues of living subjects. In years 1 and 2, we will focus primarily on method development, establishing techniques for the optimal handling and storage of blood, as well as comparing the available methodology for NAD(P)(H) measurement. In years 3-5, we will conduct a human clinical trial, examining NAD(P)(H) levels in tissues and blood, along with catabolite levels in serum and urine before and after an intervention with nicotinamide riboside, thought to increase whole blood and tissue NAD levels. By performing all of these assays in the same individuals, we will also provide the first comprehensive assessment of the value of accessible bodily fluids for predicting tissue NAD levels and, more broadly, for detecting physiologically relevant alterations in human NAD metabolism. .