Technical Abstract: Seven, antigenically distinct botulinum neurotoxins (BoNT) are produced by the bacterium Clostridium botulinum and classified into serotypes designated A-G. In animals these potent toxin acts to inhibit acetylcholine release, resulting in paralysis and death. BoNT/A and /B, together represent >80% of naturally occurring illness that is often the result of improper food storage. The extreme potency of these toxins implicates these as potential bioterrorism agents necessitating rapid and simple detection strategies for emergency responders to ensure public health. Current standards used for BoNT detection employ a mouse bioassay that requires expensive equipment and expertly trained personnel. There is a need for a rapid, deployable BoNT detection device that could be used by minimally trained personnel in the event of a foodborne outbreak or a bioterrorist threat. Here, we are the first to describe the development and application of a rapid lateral flow device (LFD) that can detect and distinguish by simple visual observation between BoNT/A and BoNT/B neurotoxins. This device can detect as little as 5 ng/mL of purified BoNT/A and 10 ng/mL of BoNT/B in milk and juice products. The LFD described here is inexpensive, sensitive, easy to use, requires no specialized training or equipment, can visually identify and distinguish between BoNT/A and /B serotypes in <20min. These attributes make this rapid diagnostic device a potentially valuable tool in the fields of food safety and homeland security.