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The development of neutralizing antibodies (NAbs) against interferon-β (IFNβ) reduces clinical efficacy and markers of bioactivity in patients with multiple sclerosis (MS), although it has also been shown that a poor response to IFNβ coincided with unexpectedly low NAb levels. To try and resolve this incoherency, this study investigated 2822 patients referred to a NAb testing facility. The reason for NAb testing was indicated for 2506 patients: routine testing (76%), worsening of disease (14%) and other reasons (10%). Overall, 31% of patients were NAb positive and 17% had titres high enough to obliterate IFNβ bioactivity. The frequency of NAbs was similar in patients in the routine testing group compared with the worsening group. Samples showing high titres failed to be associated with worsening of symptoms. The study failed to show low NAb levels in patients responding poorly to IFNβ. It is concluded that it is not possible to predict NAb status by clinical impression of treatment response. This is likely to be an effect of the partial efficacy of IFNβ. Thus routine testing for NAbs must be carried out in order to identify NAb status in patients with MS.