AbstractSince its emergence in late 2019, coronavirus disease 2019 (COVID‐19) has caused millions of deaths and socioeconomic losses. Although vaccination significantly reduced disease mortality, it has been shown that protection wanes over time, and that severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) variants of concern (VOCs) may escape vaccine‐derived immunity. Therefore, serological studies are necessary to assess protection in the population and guide vaccine regimens. A common measure of protective immunity is the presence of neutralizing antibodies (nAbs). However, the gold standard for measuring nAbs (plaque reduction neutralization test, or PRNT) is laborious and time‐consuming, limiting its large‐scale applicability. We developed a high‐throughput fluorescence reduction neutralization assay (FRNA) to detect SARS‐CoV‐2 nAbs. Because the assay relies on immunostaining, we developed and characterized monoclonal antibodies (mAbs) to lower costs and reduce the assay's vulnerability to reagent shortages. Using samples of individuals vaccinated with COVID‐19 and unvaccinated/pre‐pandemic samples, we showed that FRNA results using commercial and in‐house mAbs strongly correlated with those of the PRNT method while providing results in 70% less time. In addition to providing a fast, reliable, and high‐throughput alternative for measuring nAbs, the FRNA can be easily customized to assess SARS‐CoV‐2 VOCs. Additionally, the mAb we produced was able to detect SARS‐CoV‐2 in pulmonary tissues by immunohistochemistry assays.