NAD+ is an abundant cellular metabolite which plays vital roles in central metabolism while serving as a cofactor for oxidoreductases and cosubstrate for sirtuins and poly(ADP-ribose)polymerases (PARPs). Decreased tissue NAD+ levels have been linked to aging-associated metabolic decline and a host of chronic diseases. Cellular steady-state NAD+ levels are governed by contemporaneous synthetic and consumptive processes. Hence, lower NAD+ levels in aged tissues can arise from decreased synthesis or increased consumption. A static snapshot of the tissue levels of NAD+ is inadequate for assessing the highly dynamic pathway network which mediates NAD+ synthesis and consumption. Metabolic pathway tracing with stable isotope-labeled NAD+ precursors (e.g., nicotinamide (NAM), nicotinic acid (NA), tryptophan) and high-resolution mass spectrometry (HRMS) can unveil the individual contributions of synthesis and consumption to the steady-state NAD+ concentration. The metabolic fate of the NAD+ precursor can also be traced to metabolic products of NAD+ including NADH, NADP, and NADPH as well as intermediates in the various NAD+ biosynthetic pathways. Metabolic tracing of NAD+ synthesis and degradation as well as conversion of NAD+ to its downstream products is a highly versatile technique. It can be used to interrogate isolated cells, tissues slices, or specimens collected from preclinical or clinical in vivo studies (e.g., blood, urine, tissues). Bold claims about the pivotal role of NAD+ in human health and disease are typically fraught with uncertainty due to an incomplete understanding of NAD+ metabolism. Insight gleaned from metabolic pathway tracing can shed important new light on NAD+ metabolism and help to critically evaluate the intriguing link between cellular NAD+ levels and healthy aging.