|Botulinum neurotoxins (BoNTs) are one of the most important toxins in microbial forensics. As one of the top list of biological threats, botulinum neurotoxins (BoNTs) are the most potent natural toxins known to human. The three dimensional structures of BoNTs of types A, B, and E have been determined and revealed the three distinct structural and functional domains. The binding domain, which is responsible for cellular receptor-binding function, is located in the C-terminal portion of the heavy chain (HC). The N-terminal portion of the heavy chain (HN) comprises the translocation domain, which is hypothesized the conformational change at low pH and promote the escape of light chain from endosome. The light chain containing the catalytic motif is able to cleave different protein factors, which are attached to the protein receptors, and to block the neuromuscular transmission. Mass spectrometry is one of the key techniques in detection and identification of BoNTs. Using bioanalytical and biophysical methodologies, especially proteomics via mass spectrometry, can assess the âBoNT signaturesâ. The database of âBoNT signaturesâ will provide insightful information in detection and identification of BoNTs in microbial forensics to combat bioterrorism and biocrimes.