© 2017 EDIZIONI MINERVA MEDICA.Muscle overactivity (spasticity, dystonia or spasm) seen in certain neuromuscular disorders has been effectively treated with intramuscular injection of botulinum neurotoxins (BTXs). Since they act in the nerve terminals, toxins must be transported to the neuromuscular junctions which are generally clustered in one or more restricted areas (innervations zone(s) (IZs)) in a skeletal muscle. Likewise, IZ targeted BTX injections using guidance is highly recommended to achieve an optimal therapeutic goal with lower doses and fewer side effects. Hence, detection of the injection sites should be based on the knowledge about the localization of the IZs and also the transport mechanism of BTX in skeletal muscle is crucial for intramuscolar application of BTX. In this paper, IZ(s) of the skeletal muscles, distribution of BTX, proper sites and guidance for the injections are discussed taking into account the muscle structure and architecture.