Botulinum neurotoxins (BoNTs) are the most potent human toxins known and the 
causative agent of botulism, and are widely used as valuable pharmaceuticals. 
The BoNTs are modular proteins consisting of a heavy chain and a light chain 
linked by a disulfide bond. Intoxication of neuronal cells by BoNTs is a 
multi-step process including specific cell binding, endocytosis, conformational 
change in the endosome, translocation of the enzymatic light chain into the 
cells cytosol, and SNARE target cleavage. The quantitative and reliable potency 
determination of fully functional BoNTs produced as active pharmaceutical 
ingredient (API) requires an assay that considers all steps in the intoxication 
pathway. The in vivo mouse bioassay has for years been the 'gold standard' assay 
used for this purpose, but it requires the use of large numbers of mice and thus 
causes associated costs and ethical concerns. Cell-based assays are currently 
the only in vitro alternative that detect fully functional BoNTs in a single 
assay and have been utilized for years for research purposes. Within the last 
5 years, several cell-based BoNT detection assays have been developed that are 
able to quantitatively determine BoNT potency with similar or greater 
sensitivity than the mouse bioassay. These assays now offer an alternative 
method for BoNT potency determination. Such quantitative and reliable BoNT 
potency determination is a crucial step in basic research, in the development of 
pharmaceutical BoNTs, and in the quantitative detection of neutralizing 
antibodies.