Disease activity is highly variable in patients with multiple sclerosis (MS), both untreated and during interferon (IFN)-beta therapy. Breakthrough disease is often regarded as treatment failure; however, apart from neutralizing antibodies (NAbs), no blood biomarkers have been established as reliable indicators of treatment response, despite substantial, biologically measurable effects. We studied the biologic response to treatment in a cohort of NAb-negative patients to test whether difference in responsiveness could segregate patients with and without breakthrough disease during therapy.