Multiple sclerosis (MS) is thought to correlate with an array of clinically relevant biomarkers produced during inflammatory process. In this study, a novel gene expression profiling technology was developed and characterized to quantitatively measure the expression profiles of 34 genes selected based on their role in inflammation and their susceptibility to regulation by current MS treatment agents, beta-interferon (IFN) and glatiramer acetate (GA). Potential clinical applications of the technology were evaluated by in vitro and ex vivo analyses in peripheral blood mononuclear cells (PBMC) obtained from MS patients and controls. Interferon-inducible genes were universally up-regulated after in vitro treatment with beta-IFN while the expression of other selected genes encoding cytokines and molecules related to T cell trafficking, activation and apoptosis was variably affected. Beta-IFN and GA exhibited distinctive and characteristic regulatory effects on the expression of the selected genes. Similar regulatory properties of beta-IFN and GA were seen by ex vivo analysis of PBMC specimens in a self-paired study by comparing specific changes induced by beta-IFN or GA treatment in the same patients as well as in a group study by measuring specific profiles in treatment groups compared with an untreated group. Furthermore, the technology served as a simple and sensitive assay for detection of beta-IFN neutralizing antibody based on the blocking effect of serum antibodies on the known regulatory properties of beta-IFN on PBMC. The findings provide important information on the immunoregulatory properties of beta-IFN and GA and support potential clinical applications of this technology in detection of neutralizing antibody (NAB) and evaluation of treatment responses in MS patients.