Neutralizing monoclonal antibodies and nanobodies have shown promising results as potential therapeutic agents for COVID-19. Identifying such antibodies and nanobodies requires evaluating the neutralization activity of a large number of lead molecules via biological assays, such as the virus neutralization test (VNT). These assays are typically time-consuming and demanding on-lab facilities. Here, we present a rapid and quantitative assay that evaluates the neutralizing efficacy of an antibody or nanobody within 1.5 h, does not require BSL-2 facilities, and consumes only 8 μL of a low concentration (ng/mL) sample for each assay run. We tested the human angiotensin-converting enzyme 2 (ACE2) binding inhibition efficacy of seven antibodies and eight nanobodies and verified that the IC50 values of our assay are comparable with those from SARS-CoV-2 pseudovirus neutralization tests. We also found that our assay could evaluate the neutralizing efficacy against three widespread SARS-CoV-2 variants. We observed increased affinity of these variants for ACE2, including the β and γ variants. Finally, we demonstrated that our assay enables the rapid identification of an immune-evasive mutation of the SARS-CoV-2 spike protein, utilizing a set of nanobodies with known binding epitopes.