Soil tests can help optimize nitrogen (N) fertilizer rates, thereby improving farmer profitability and environmental performance. In US Midwest maize (Zea mays) production, however, most soil N tests have limited accuracy to predict N fertilizer requirements. Here we tested the individual and combined ability of 30 soil tests (12 rapid N extractions, seven biological carbon or N tests, six long-term incubation kinetic parameters, and five other routine soil tests), as well as environmental and management data, to predict maize response to N fertilizer across 56 site-years in the US Midwest. Out of 30 soil tests, and across all site-years, a 14-d aerobic incubation best predicted whether maize responded to N fertilizer, and a 5-min tetraphenyl borate extraction best predicted agronomic optimum N rate. We combined these two tests to evaluate their ability to predict N fertilizer response against the most commonly used soil N test in the US Midwest, the pre-sidedress or late-spring nitrate test (PSNT or LSNT). The combination of soil tests nearly doubled the ability to predict nonresponsive sites compared to PSNT, and on average resulted in a 40% reduction in over-application and 37% reduction in under-application of N fertilizer. Weather and management variables marginally improved the prediction of maize N response.Our results indicate that a simple combination of biological N mineralization (14-d aerobic incubation) and chemical extraction (5-min tetraphenyl borate) assays could improve current N fertilizer recommendations.Abbreviations: AEIM, 14-day aerobic incubation with moist soils; AONR, agronomic optimum nitrogen rate; KCl8, 8-minute 2 M KCl extract for nitrate; LSNT, late-spring nitrate test; NRHY, maize nonresponsive to fertilizer nitrogen but high-yielding site-year; NRLY, maize nonresponsive to fertilizer nitrogen but low-yielding site-year; NUE, nitrogen use efficiency; PSNT, pre-sidedress nitrate test; RN, maize responds to nitrogen fertilizer; SOC, soil organic carbon; STK, Mehlich III extractable potassium; STP, Mehlich III extractable phosphorus; TPB5, 5-minute tetraphenyl borate extraction of ammonium.This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.