Neutralizing antibodies (NAbs) can occur in some multiple sclerosis (MS) patients receiving interferon beta (IFNbeta) therapy. NAbs reduce drug bioavailabity and high NAb titers reduce drug efficacy. We describe the validation of the R. Farrell and G. Giovannoni luciferase reporter gene assay to measure NAbs to INFbeta. We assayed 163 sera from IFNbeta treated MS patients with an optimized luciferase method and compared the results to those obtained with the reference cytopathic effect (CPE) method using A549 cells and an encephalomyocarditis virus (EMCV). Binding antibodies (BAbs) were measured using a capture ELISA as a screening test for NAbs in the CPE assay. NAb status measured by the luciferase and the ELISA/CPE method did not yield a significant difference. Log10 NAb titers obtained from the luciferase assay and the A549/EMCV CPE methods correlated very well. The inter-assay coefficient of variation for titers was between 17.8-29.3%, and the intra-assay coefficient of variation was between 6.3-15.2%. The luciferase assay is reliable, appropriately sensitive and requires less time than the currently available NAb methods.