Botulinum toxins, some of the most poisonous naturally occurring substances are proteins produced by the bacterinum Clostridium botulinum. They are neurotoxic, that is toxic to the nerve cells and responsible for causing botulism, most often associated with eating food containing toxin or in hard drug users. The toxins cause respiratory and muscle paralysis, and even death, by blocking the nerve function. Treatment is possible with antitoxins, provided they are given to patients on time. Although highly toxic in extremely small quantities toxin can be administered safety to treat painful muscle spasms and involuntary muscle contractions. It is increasingly applied to many new medical conditions and for cosmetic purposes. Botulism is diagnosed by injecting animals with body fluids from patients and in suspected food samples to confirm presence of toxins. In pharmaceutical industries manufacturers of therapeutic toxins and antitoxins require many animals in severe lethality assay to confirm safety, potency and stability. Alternative methods developed to date have limitations by either still relying on animal or for provision of tissues or for reflecting only one of several factors that contribute to toxin mode of action in animals. One approach to avoid these limitations is to develop in vitro functional assay which offers the potential for entire animal replacement because of involving all key steps of botulinum intoxication. Because botulinum toxin induces paralysis by blocking the release of chemicals (neurotransmitters such as acetylcholine) at the neuromuscular nerve endings, relevant in vitro assays must focus on this activity. Established human neuronal cell lines and human stem cells have not offered the required sensitivity and studies to date have confirmed need for differentiation into neuronal like structures before these cells can be used with neurotoxins. In this work we will apply differentiated cells onto micro-electrode arrays (MEAs) in order to perform measurement of cell function in situ. We will support these studied by also looking at cell recycling activity by staining of the components involved in neurotransmitter release and by selectively detecting key proteins within the cells which are attacked by toxins and are also associated with neurotransmitter release. This new approach reflects the full function of the toxin yet it is entirely non animal orientated and will not rely on any animal experimentation used in other applications and strategies.