Interferon beta (IFNβ) therapy has immunogenic properties and induces the development of neutralizing antibodies (NAbs). From the extensive literature focused in the development of NAbs in multiple sclerosis (MS) patients, their ability to cross-react has been deficiently evaluated, despite having important consequences in the clinical practice. Here, the relation between the cross-reactivity and the NAbs titers has been evaluated in MS patients, by inhibition of the antiviral activity of IFNβ by bioassay and through the interference with the activation of the IFNß pathway (JAK-STAT), by phosphoflow. Thus, patients with intermediate-high titers of NAbs, determined by bioassay, had a 79-fold increased risk of cross-reactivity compared to patients with low titers. The cross-reactivity is also demonstrated because NAbs positive sera were able to decrease significantly the activation of pSTAT1 achieved by other different IFNβ molecules in the cells patients. Besides, a linear relationship between the STAT1 phosphorylation and NAbs titers was found. The study demonstrates that cross-reactivity increases with the titer of antibodies, which has important implications in clinical practice when switching the treatment. The direct relationship between the NAbs titer and the activation of STAT1 suggest that its determination could be an indirect method to identify the presence of NAbs.