Nicotinamide adenine dinucleotide (NAD) is an important cofactor of energy-producing pathways. The redox ratio (NAD ⁺ /NADH) reflects the cellular oxidoreductive state. Oxidative stress and redox dysregulation have been suggested to contribute to various neurological diseases. The assessment of NAD content has been recently demonstrated in large animals and human brains by ³¹ P magnetic resonance spectroscopy. However, its measurement in small rodents has never been attempted. The purpose of this study was to investigate, in vivo, the NAD content during mouse brain neurodevelopment. ³¹ P-MR-spectra were acquired in the mouse brain at postnatal days P20, P40, P90 and P250 at 14.1 T using a 3D-localization sequence. High spectral quality was achieved at 14.1 T. NAD ⁺ and NADH were quantified with mean Cramér-Rao lower bound of 10% and 14%, respectively. An increase in NAD ⁺ /NADH was observed from P20 to P250 due to a decrease in [NADH]. The intracellular pH was significantly reduced with age, while the free [Mg ²⁺ ] in the brain was significantly increased. This study demonstrates for the first time the feasibility of the measurement of NAD content in vivo in mouse brains during development, which opens the prospect of longitudinally studying energy metabolism and redox dysfunction in mouse models of brain pathology.