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Since its emergence in late 2019, infection by SARS-CoV-2 (COVID-19 disease) has quickly spread worldwide, leading to a pandemic that has caused millions of deaths and huge socio-economic losses. Although vaccination against COVID-19 has significantly reduced disease mortality, it has been shown that protection wanes over time, and that circulating SARS-CoV-2 variants may escape vaccine-derived immunity. Therefore, serological studies are still necessary to assess protection in the population and better guide vaccine booster programs. A common measure of protective immunity is the presence of neutralizing antibodies (nAbs). However, the gold standard method for measuring nAbs (plaque reduction neutralization test, or PRNT) is laborious and time-consuming, limiting its large-scale applicability. In this study, we developed a high-throughput fluorescence reduction neutralization assay (FRNA) to detect SARS-CoV-2 nAbs. Because the assay relies on immunostaining, we also developed and characterized in-house monoclonal antibodies (mAbs) to lower assay costs and reduce the vulnerability of the test to reagent shortages. Using samples collected before the pandemic and from individuals vaccinated against COVID-19, we showed that the results of the FRNA we developed using commercial and in-house mAbs strongly correlated with those of the standard 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 other SARS-CoV-2 variants of concern (VOCs). We also demonstrated the applicability of the mAbs produced in immunohistochemistry assays.