Nitrogen is one of the crucial macronutrients essential for plant growth, development, and survival under stress conditions. Depending on cellular requirement, plants can absorb nitrogen mainly in multiple forms such as nitrate (NO₃^-) or ammonium (NH₄⁺) or combination of both via efficient and highly regulated transport systems in roots. In addition, nitrogen-fixing symbiotic bacteria can fix atmospheric nitrogen in to NH₄⁺ via highly regulated complex enzyme system and supply to the roots in nodules of several species of leguminous plants. If NO₃^- is a primary source, it is transported from roots and then it is rapidly converted to nitrite (NO₂^-) by nitrate reductase (NR) (EC 1.6.6.1) which is a critical and very important enzyme for this conversion. This key reaction is mediated by transfer of two electrons from NAD(P)H to NO₃^-. This occurs via the three redox centers comprised of two prosthetic groups (FAD and heme) and a MoCo cofactor. NR activity is greatly influenced by factors such as developmental stage and various stress conditions such as hypoxia, salinity and pathogen infection etc. In addition, light/dark dynamics plays crucial role in modulating NR activity. NR activity can be easily detected by measuring the conversion of NO₃^- to NO₂^- under optimized conditions. Here, we describe a detailed protocol for measuring relative NR enzyme activity of tomato crude extracts. This protocol offers an efficient and straightforward procedure to compare the NR activity of various plants under different conditions.