Nitrate assimilation in the plant cell is mainly regulated by the enzyme nitrate reductase (EC 1.6.6.1), which catalyzes the nitrate to nitrite reduction. Nitrate reductase activity (NRA) is measured at the major nitrate reduction site, which can be the root or shoot, depending on the species. The in vivo assay has often been used for NRA measurement, and protocol also usually varies with the species. The goals of this study were: (1) to identify the major nitrate reduction site in seedlings of two tree species, Eucalyptus urophylla and Khaya senegalensis, and (2) to optimize the in vivo nitrate reductase assay at the major nitrate reduction site in these species. Healthy seedlings aged 180 and 160 days, respectively, were selected for NRA measurement in fully expanded leaves and main root. After identifying the main nitrate reduction site of each species, the effects of variations in temperature, nitrate concentration and pH in the incubation medium were assessed. The results showed that the leaf and the root are the major nitrate reduction site of Eucalyptus urophylla and Khaya senegalensis, respectively. The optimal conditions for the in vivo assay in the leaf were 35 °C, KNO3 100 mM, and pH 7.0, whereas for the root they were 30 °C, KNO3 100 mM, and pH 7.5.