Botulinum neurotoxins are the most lethal naturally produced neurotoxins. Due to the extreme toxicity, botulinum neurotoxins are implicated in bioterrorism; while, the specific mechanism of action and the long-lasting effect were found to be medically applicable in treating various neurological disorders. Developing assays that can accurately determine toxicity of botulinum neurotoxins is therefore highly desirable to protect the public from bioterror threats and also to ensure the safety of patients on therapies derived from botulinum neurotoxins. In the first part of the thesis, I discuss different approaches that I’ve taken to develop physiologically relevant cell-based assays including (i) coculture of Schwann cells and neuronal cells, (ii) optimization of neural cell culture media, and (iii) development of an organotypic neuromuscular junction-like model to mimic muscle paralysis in vitro. Additionally, I explored an alternative therapeutic application of botulinum neurotoxins. Synaptic vesicle 2 proteins are receptors of botulinum neurotoxin type A, which are highly expressed in neuroendocrine tumors and have been recognized as new biomarkers. I hypothesized that the nontoxic receptor-binding domain of botulinum neurotoxin type A can be used as a ligand to target synaptic vesicle 2 proteins expressed in neuroendocrine tumors without imposing neurotoxicity. In the latter part of the thesis, I show that the nontoxic fragment of the heavy-chain receptor binding domain of botulinum neurotoxin type A preferentially recognizes and internalizes into neuroendocrine cancer cells originating from thyroid, lung, and pancreas. More intriguingly, this heavy-chain receptor binding domain does not only target neuroendocrine cancer cells but also suppresses expression of chromogranin A and achaete-scute complex 1, neuroendocrine tumor markers, indicating potential application in palliative therapy and reversion of neuroendocrine differentiation.