Technical Abstract: The gold standard assay for measuring the activity and typing of Clostridium botulinum neurotoxins is the mouse bioassay. The mouse bioassay is sensitive, robust and does not require specialized equipment. However, the mouse bioassay is slow, not practical for many settings and results in the death of animals. Here we describe an in vitro SNAP-25 cleavage assay for measuring the toxin activity with the sensitivity of the mouse bioassay. Moreover, this assay is far more rapid, can be automated and adapted to many lab settings: and has the potential to be used for toxin typing. The assay has two main steps, the first is immunoseparation and concentration of the toxin using immunomagnetic beads with monoclonal antibodies directed against the 100 kDa heavy chain subunit, and the second is a cleavage assay targeting the SNAP-25 peptide of the toxin, labeled with fluorescent dyes and detected as a FRET assay. Our results suggest that the sensitivity of this assay is 10 pg/ml, which is similar to the sensitivity of the mouse bioassay, and can detect the activity of the toxin in various foods. These results suggest that the assay has a potential use as an alternative to the mouse bioassay, for analysis of Clostridium botulinum type A neurotoxin.