Nicotinamide adenine dinucleotide (NAD(+)) is a central metabolic coenzyme/cosubstrate involved in cellular energy metabolism and energy production. It can readily be reduced by two electron equivalents and forms the NADH form, which is the minority species to NAD(+) under most physiologic conditions. NAD(+) plays an important role in not only oxidation-reduction reactions in cells but also as a signaling molecule. For example, NAD(+) plays a key role in mitochondrial function via participation in pyruvate dehydrogenase, tricarboxylic acid cycle, and oxidative phosphorylation chemistries. It also serves as a substrate for deacylases SIRT3, SIRT4, and SIRT5, which modify protein posttranslational modifications on lysine within the mitochondrial compartment. Recent work has highlighted the biological significance of dynamic changes to mitochondrial NAD(+). This has increased the need for standardized and effective methods to measure NAD(+) contents in this organelle. To determine NAD(+) concentrations in cells, and specifically in mitochondria, we describe two assays for NAD(+) determinations: An Enzymatic Cycling Assay and Isotope Dilution. The cycling assay contains sample NAD(+), lactate, lactate dehydrogenase, diaphorase, and resazurin. The isotope dilution assay uses synthetic (18)O-NAD(+) as an internal standard, and treated samples are fractionated by HPLC and then NAD(+) concentration determined by the (16)O- and (18)O-NAD(+) peak (664/666) ratio in positive mode MS.