Using 15N stable isotopes to quantify nitrogen (N) transformation rates has been proven to be a powerful approach for increasing understanding of N cycles in various ecosystems because they can contribute both source-sink and process information. One major challenge in methods for determination of 15NO3– concentrations is reducing the test cost via replacing expensive equipment and at the same time decreasing the pretreatment times and sample volumes. Here we describe a novel method (Zn-OX/MIMS) of determining 15NO3– concentrations for isotope-enrichment experiments via membrane inlet mass spectrometer (MIMS). We reduce dissolved 15NO3– to 15NH4+ using zinc powder under an optimized condition based on lots of preliminary experiments and oxidize 15NH4+ to 29N2 and/or 30N2 completely by hypobromite iodine solution, and then analyze produced gases with MIMS. The Zn-OX/MIMS method provides an accurate and precise approach to quantify 15NO3– concentrations in water samples over a range of salinities (R2 ≥ 0.9997, p < 0.0001) and a 2 M KCl solution (R2 = 0.9996, p < 0.0001). For applications, this method provides a convenient way to measure the gross nitrification and 15NO3– immobilization rates by isotope dilution in various ecosystems. In addition, using Zn-OX/MIMS method for determination of 15NO3– concentrations in aqueous samples can significantly simplify equipment and experimental procedure, and decrease the pretreatment times, cost, and sample volumes. Therefore, this method will promote the application of MIMS in N cycle research and contribute enormously to our understanding of N transformations, fate, and overall dynamics in numerous ecosystems in the future.