The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a ‘Serological Assay based on a Tri-part split-NanoLuc® (SATiN)’ to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Herein, we expand on our previous work and describe a reconfigured version of the SATiN assay that can measure neutralization activity of antibodies directly from convalescent or vaccinated sera. The sensitivity is comparable to cell-based pseudovirus neutralization assays but with significantly shorter preparation and assay run time. As the assay is modular, we further demonstrate that Neutralization SATiN (Neu-SATiN) enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.