We measured the frequency of binding antibody (BAB) and neutralizing antibody (NAB) against interferon-beta (IFN-beta) in sera from 827 patients with relapsing-remitting multiple sclerosis (RRMS) using various IFN-beta medications. Of the 827 patients, 363 (43.9%) had serum BAB after more than 12 months of IFN-beta treatment. Of the 363 BAB-positive sera, we analyzed 137 for BAB using a myxovirus-resistant protein A (MxA) protein induction assay (ELISA) and an MxA messenger RNA (mRNA) induction assay (real-time polymerase chain reaction (PCR)). We obtained similar results for each serum in both NAB assays, indicating a good correlation between transcription and translation of MxA. We tested all 363 sera by real-time PCR, and NAB was present in 70.8% of BAB-positive sera, or 31.1% of the 827 patients with relapsing-remitting multiple sclerosis (RRMS). Real-time PCR offers several advantages for measuring NAB compared with the MxA protein induction enzyme-linked immunosorbent assay and should be used routinely for NAB among patients with MS using IFN-beta treatment.