Interferon-Beta (IFNβ) is a cytokine, or glycoprotein, which is released by virtually all cell subsets in response to viral and bacterial pathogens. It has been used as a therapy to suppress multiple sclerosis (MS) disease activity for approximately 20 years. The purpose of this study was to characterize the role of endogenous, or internally produced, IFNβ in the development of the autoimmune response in this disease by evaluating its biological activity in the serum of patients with relapsing-remitting (RR) MS in comparison to healthy controls (HC). The results of a sensitive cell-based bioassay showed reduced expression of the interferon-inducible genes, myxovirus resistance 1 (MX1) and protein kinase RNA regulated protein (PRKR), in RRMS serum-treated WISH cells, suggesting a deficient endogenous IFNβ biological activity in RRMS patients in comparison to HCs. This study will enable physicians to identify patients with lower endogenous IFNβ levels who will optimally respond to IFNβ-1a treatment.