We have recently developed several small molecule inhibitors of Botulinum Neurotoxin (BoNT) using a fragment-based approach guided by structural data and computer-aided lead optimization. In vitro assay and cell-based assays showed that these are the most potent inhibitors described against BoNT to date. We are now evaluating the safety and efficacy of the lead inhibitors in a small animal model of BoNT inhalational exposure. Inhalation is the most likely form of delivery of BoNT if it is used as a biological weapon. Emerging data from other groups reveal severe impairment of several respiratory functional parameters following BoNT/A exposure and lack of protection by the pentavalent toxoid against pulmonary injury. We are adopting our microinstillation technology, which involves aerosolizing the toxin in the trachea using a microcatheter to develop a BoNT inhalation exposure model. The simplicity of the procedure and its requirement of only microliters of a toxin to generate a meaningful and reliable dose–response suggest that it may be a suitable method for acute inhalation toxicity evaluation of BoNT aerosol. Post-exposure respiratory functional analysis by plethysmography may identify sensitive non-invasive parameters for rapid and early diagnosis of BoNT exposure. Since the airway and lung is the primary target for inhaled BoNT and the lung pathology persists, development of a small animal inhalation model and evaluation of potent small molecule BoNT inhibitors is important to develop therapeutics to protect against both the pulmonary pathology as well as systemic toxicity in the event of an aerosol release.