More than 16 years ago human interferon-β-1b (IFN-β-1β) was shown to be effective in the treatment of the relapsing-remitting form of multiple sclerosis (MS). Over time, IFN-β has been demonstrated to be both a safe and effective treatment. However, the mechanism of action of IFN-β in MS remains unknown. To better understand the mechanism of action of IFN-β, considerable effort has been made in transcriptional profiling of peripheral blood mononuclear cells collected from MS patients. IFN-β is known to induce a large number of genes that play an important role in regulating responses to viral infection, immune modulation, and cell proliferation. Identifying differentially induced genes that are linked to the beneficial effects observed during treatment is under active investigation. IFN biomarkers in MS patients have been proposed but have not been clearly confirmed in independent studies or consistently correlated with clinical measures of disease progression. Organizing single genes or gene signatures grouped according to molecular mechanisms meaningful in MS may help to link IFN activity measurements to clinical outcomes. In this review, IFN activity measurements will be discussed with a specific emphasis on what is known about differential gene expression and treatment effects in MS.