Multiple sclerosis is an autoimmune disease of the central nervous system believed to be mediated by pathogenic T lymphocytes. We have developed a next-generation therapy in which cells secrete specific therapeutic molecules to silence these aberrant T cells. We have shown that fibroblasts, transduced to secrete a myelin basic protein-derived peptide, abrogate disease in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis, which we hypothesized using a low-zone tolerance mechanism. To determine the efficacy (or not) of this therapy in humans, we must ensure that patients receive comparable doses of therapeutic peptide. To this end, we have used liquid chromatography coupled to tandem mass spectrometry to detect a tryptic peptide, derived from the secreted therapeutic product, at nanomolar concentrations. Success depended on growing the transduced fibroblasts in defined PC-1 medium in the presence of a cocktail of protease inhibitors.