Technical Abstract: Current methods for detecting the presence of botulinum neurotoxins in food and other biological samples Botulinum neurotoxins (BoNTs), the causative agents of botulism, are among the most lethal human bacterial toxins and the causative agent of botulism. BoNTs are also classified as Select Agents and have been used as agents of bioterrorism. Usually an identification of botulism is made through clinical manifestations and diagnosis, with subsequent confirmation by laboratory identification of clostridial spores or toxin in food, environmental, or clinical samples. The speed of recovery from botulism increases with the timely administration of antitoxin or medical interventions. Thus, sensitive and rapid toxin detection and diagnostic methods are critical for improved recovery time as well as facilitate the epidemiologic study of contamination and forensic identification of outbreaks. A multitude of assay formats have been developed over many years, with in some cases, reported sensitivities at the attomolar level. Many such assays are not usable for the detection of BoNT contamination in food or other complex biological samples. This book chapter focuses on the diagnostic methods for toxin detection and the challenges encountered in adapting analytical methods for detection of BoNTs in foods and other biological and environmental samples. The chapter will briefly describe the properties of BoNTs as they relate to detection methods, will compare and contrast methods currently in use for food and biological sample analyses, and discuss new methods under development.