Activity of nitrate reductase (NR), the first enzyme in the nitrate-assimilation pathway, was estimated in the cotyledons of the sunflower (Helianthus annuus) using a standardized in-vivo method. Seedlings were grown in the light on a nitrate medium. Various factors that affect NR activity were optimized, including the molarity and pH of the reaction buffer, nitrate concentration, and use of a surfactant. We also determined whether NADH was required for nitrate reduction. The surfactant propanol (2%) gave the best results, and no NADH supplement was necessary. In a separate study, we compared the effect of various culturing components on in-vivo NR activity among monocot and dicot species, and found that Triton X-100 was the best surfactant for monocots whereas dicots performed better with n-propanol. Monocot species also required additional NADH as an external energy source. Moreover, specific purification procedures were needed to enhance NR activity in dicotyledons. Finally, we also assessed the efficacy of in-vivo versus in-vitro procedures for assaying monocots versus dicots.