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There are numerous methods for measuring inorganic nitrogen (N) in soils, but many of these are complex or require expensive equipment. In order to seek an accurate and rapid measurement method, we optimized the conditions for soil inorganic nitrogen (N) (NH4+-N and NO3–N) determination with a micro-plate reader; a systematic study was carried out. The effects of extraction regent concentrations, ratios of extraction solution to the soil, extraction time, extracted solution storage methods and time, and reaction time on soil inorganic N measurement were investigated. Results showed that all tested conditions except the storage method had a significant effect on the determination of soil NH4+-N and NO3–N. Compared with the non-stored treatment, the value of the measured soil nitrate-nitrogen increased after being stored in a refrigerator for seven weeks, while that for ammonium-nitrogen content was reduced. The appropriate protocol was using extracting solution directly to determine the content of NH4+-N and NO3–N in soil (otherwise, keep it at room temperature), 1.0 mol/L KCl as the extraction regent, solution/soil ratio with 10:1, extraction for 45 min, and reaction for 25 min (only for NH4+-N). The recovery rate of adding standard solution was above 99% as it met the detection requirements.