A comprehensive understanding of neural distribution within the vastus medialis is crucial for the effective administration of botulinum neurotoxin injections to manage spasticity. The aim of this study was to develop an anatomically informed approach to guide the administration of botulinum neurotoxin injections into the vastus medialis muscle. Using a modified Sihler's method, we examined the vastus medialis muscles (20 specimens) to delineate the distribution of nerves relative to a transverse line extending from the anterior superior iliac spine to the base of patella. The vastus medialis muscle was divided into 10 areas from top to bottom. Then, using two fresh cadavers, ultrasonography-guided injections were performed based on the distribution of nerves within the vastus medialis. Each specimen was subsequently dissected to verify if the dye was accurately directed to the most densely innervated regions of the vastus medialis and to assess the precision of the injections. The intramuscular nerve distribution within the vastus medialis muscle showed distinct patterns, particularly in areas between 6 and 9. Four injections were successfully administered on each side, targeting the regions between 6 and 9 of the vastus medialis. Upon dissection of the cadavers, the dye was found to be distributed along the muscle fiber. We recommend targeting botulinum neurotoxin injections toward regions displaying a prominent nerve distribution, specifically focusing on areas between 6 and 9. By adhering to these guidelines, clinicians can minimize doses and mitigate potential adverse effects, such as gait disturbances, antibody development, and bruising, resulting from multiple injections. Furthermore, these findings can be incorporated into electromyography practices.