Botulinum neurotoxin (BoNT), which cause the deadly neuroparalytic disease, botulism, is the most toxic substance known to man. BoNT can be used as potential bioterrorism agents, and therefore, pose great threat to national security and public health. Rapid and sensitive detection of BoNTs using molecular and biochemical techniques is an essential component in the diagnosis of botulism, and is yet to be achieved. The most sensitive and widely accepted assay method for BoNTs is mouse bioassay, which takes 4 days to complete. This clearly can not meet the need for clinical diagnosis of botulism, botulinum detection in field conditions, and screening of large scale samples. Consequently, the clinical diagnosis of botulism relies on the clinical symptom development, thus limiting the effectiveness of antitoxin treatment. In response to this critical need, many in vitro methods for BoNT detection are under development. This review is focused on recently developed in vitro detection methods for BoNTs, and emerging new technologies with potential for sensitive and rapid in vitro diagnostics for botulism.