Antibody titers against a viral pathogen are typically measured using an antigen binding assay, such as an enzyme-linked immunosorbent assay (ELISA), which only measures the ability of antibodies to identify a viral antigen of interest. Neutralization assays measure the presence of virus-neutralizing antibodies in a sample. Traditional neutralization assays, such as the plaque reduction neutralization test (PRNT), are often difficult to use on a large scale due to being both labor and resource intensive. Here we describe an Ebola virus fluorescence reduction neutralization assay (FRNA), which tests for neutralizing antibodies, that requires only a small volume of sample in a 96-well format and is easy to automate. The readout of the FRNA is the percentage of Ebola virus-infected cells measured with an optical reader or overall chemiluminescence that can be generated by multiple reading platforms and the readout is compatible with lytic and non-lytic viruses. Using blinded human clinical samples (EVD survivors or contacts) obtained in Liberia during the 2013–2016 Ebola virus disease outbreak, we demonstrate that FRNA-measured antibody titers are highly correlated with those measured by the Filovirus Animal Non-clinical Group (FANG) ELISA - the current standard for anti-EBOV antibody measurement with the important distinction of providing information on the neutralizing capabilities of the antibodies.