Background: The primary sources of nitrogen for plants have been suggested to be nitrate (NO3 −) and ammonium (NH4 +). However, when both nitrate and ammonium are simultaneously available to plants, it is very difcult to diferentially quantify NO3 −/NH4 + utilization in culture media or soil. Consequently, the contribution of NO3 −/NH4 + to total inorganic nitrogen assimilation cannot be determined. Results: We developed a method called the bidirectional stable nitrogen isotope tracer to diferentially quantify the nitrate and ammonium utilization by Orychophragmus violaceus (Ov) and Brassica napus (Bn) plantlets in vitro. The utilization efciency of nitrate was markedly lower than the utilization efciency of ammonium for plantlets of both Ov and Bn. In both Ov and Bn, the proportion of NO3 −/NH4 + utilization did not show a linear relationship with inorganic nitrogen supply. The Ov plantlets assimilated more nitrate than the Bn plantlets at the lowest inorganic nitrogen concentration. Conclusions: Quantifying the utilization of nitrate and ammonium can reveal the diferences in nitrate and ammonium assimilation among plants at diferent inorganic nitrogen supply levels and provide an alternate way to conveniently optimize the supply of inorganic nitrogen in culture media.