Nitrate reductase (NR) catalyzes rate-limiting and controlled step in nitrate assimilation in higher plants. This cytoplasmic enzyme responds to light/dark signals. Regulation of NR by light being complex is manifested through gene expression at transcription and translation, covalent modification and reductant supply. Past efforts have led to an understanding of covalent modification of NR via phosphorylation/dephosphorylation mechanism. This book includes in vivo and in vitro NR assay optimization and measurements in spinach during light/dark transition in presence and absence of Mg2+ and ATP alone and their combinations. In vivo NR activity declined gradually under dark with significant decline being noticed only after 60 min. Also, in vivo activity declined slowly up to 60 min of darkness in presence of Mg2+, the decline being slow in its absence. Decline of in vitro NR activity was not observed in presence of Mg2+ up to 30 min of darkness, but the activity declined gradually in presence of ATP up to 60 min of darkness. Further, considerable decline of in vitro enzyme activity was noticed after 15 min of darkness in presence of Mg2+ ions and ATP.