Botulinum neurotoxins (BoNTs) are proteases that cleave specific cellular 
proteins essential for neurotransmitter release. Seven BoNT serotypes (A-G) 
exist; 4 usually cause human botulism (A, B, E, and F). We developed a rapid, 
mass spectrometry-based method (Endopep-MS) to detect and differentiate active 
BoNTs A, B, E, and F. This method uses the highly specific protease activity of 
the toxins with target peptides specific for each toxin serotype. The product 
peptides derived from the endopeptidase activities of BoNTs are detected by 
matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. In 
buffer, this method can detect toxin equivalents of as little as 0.01 mouse 
lethal dose (MLD)50 and concentrations as low as 0.62 MLD50/mL. A 
high-performance liquid chromatography-tandem mass spectrometry method for 
quantifying active toxin, where the amount of toxin can be correlated to the 
amount of product peptides, is also described.