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Nicotinamide adenine dinucleotide (NAD) is found in all living cells where the oxidized (NAD + ) and reduced (NADH) forms play important roles in many enzymatic reactions. However, little is known about NAD + and NADH conformational changes and kinetics as a function of the cell environment. In the present work, an analytical workflow is utilized to study NAD + and NADH dynamics as a function of the organic content in solution using fluorescence lifetime spectroscopy and in the gas-phase using trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) and infrared multiple photon dissociation (IRMPD) spectroscopy. NAD solution time decay studies showed a two-component distribution, assigned to changes from a “close” to “open” conformation with the increase of the organic content. NAD gas-phase studies using nESI-TIMS-MS displayed two ion mobility bands for NAD + protonated and sodiated species, while four and two ion mobility bands were observed for NADH protonated and sodiated species, respectively. Changes in the mobility profiles were observed for NADH as a function of the starting solution conditions and the time after desolvation, while NAD + profiles showed no dependence. IRMPD spectroscopy of NAD + and NADH protonated species in the 800–1800 and 3200–3700 cm −1 spectral regions showed common and signature bands between the NAD forms. Candidate structures were proposed for NAD + and NADH kinetically trapped intermediates of the protonated and sodiated species, based on their collision cross sections and IR profiles. Results showed that NAD + and NADH species exist in open, stack, and closed conformations and that the driving force for conformational dynamics is hydrogen bonding of the N–H–O and O–H–O forms with ribose rings.