Objective We investigated the predictive value of the enzyme-linked immunospot technique (ELISPOT) in identifying patients with relapsing-remitting multiple sclerosis (RRMS) who will respond to treatment with glatiramer acetate (GA) or interferon-β (IFN-β), based on the brain-reactive B-cell activity of peripheral blood cells. Methods In this retrospective, cross-sectional, real-world multicenter study, we identified patients with RRMS in the NeuroTransData MS registry and stratified them based on their documented treatment response (relapse-free in the first 12 months of treatment) to GA or IFN-β. The GA group comprised 73 patients who responded to GA and 35 nonresponders. The IFN-β group comprised 62 responders to IFN-β and 37 nonresponders. Patients with previous or current therapy affecting B-cell activity were excluded. We polyclonally stimulated mononuclear cells from peripheral blood samples (collected after participant selection) and investigated brain-reactive B-cell activity after incubation on brain tissue lysate-coated ELISPOT plates. Validity metrics of the ELISPOT testing results were calculated (Python 3.6.8) in relation to the clinical responsiveness in the 2 treatment groups. Results The ELISPOT B-cell activity assay showed a sensitivity of 0.74, a specificity of 0.76, a positive predictive value of 0.78, a negative predictive value of 0.28, and a diagnostic OR of 8.99 in predicting clinical response to GA vs IFN-β therapy in patients with RRMS. Conclusion Measurement of brain-reactive B-cell activity by ELISPOT provides clinically meaningful predictive probabilities of individual patients’ treatment response to GA or IFN-β. The assay has the potential to improve the selection of optimal first-line treatment for individual patients with RRMS.