Some interferon beta (IFNbeta)-treated patients with multiple sclerosis develop antibody-mediated decreased bioactivity with resultant loss of therapeutic effect. The authors developed real-time multiplex reverse transcriptase PCR to measure expression of three IFNbeta-inducible genes to directly assess IFNbeta bioactivity in patients with neutralizing antibodies (NAbs). The three genes responded in tandem. Correlation of NAb level with bioactivity at low/moderate NAb levels was poor, indicating that for such patients, direct measurement of IFNbeta bioactivity is most reliable.