Clostridium botulinum neurotoxin (BoNT) serotypes A and B are widely used as pharmaceuticals to treat various neurological disorders and in cosmetic applications. The major adverse effect of these treatments has been resistance to treatment after multiple injections. Currently, patients receiving BoNT therapies and patients enrolled in clinical trials for new applications and/or new formulations of BoNTs are not routinely monitored for the formation of neutralizing antibodies, since no assay other than the mouse protection procedure is commercially available that reliably tests for the presence of such antibodies. This report presents a highly sensitive and specific neuronal cell-based assay that provides sensitive and specific detection of neutralizing antibodies to BoNT/A.