Several key biological mechanisms of traumatic injury to axons are being elucidated using in vitro stretch injury models. However, these models are based on the experimentation of single cultures keeping productivity slow. Indeed, low yield has hindered important and well-founded investigations requiring high throughput methods such as proteomic analyses. To meet this need, we engineered a multi-well high throughput injury device to accelerate and accommodate the next generation of traumatic brain injury research. This modular system stretch-injures neuronal cultures in either a 24-well culture plate format or 6 individual wells simultaneously. Custom software control allows the user to set the input pressure and valve timing to achieve the desired substrate deformation and injury parameters. Characterization plots were created to aid the user in choosing the programmed parameters. Precise control of the pressure pulse was achieved. Peak pressure was linearly related to input pressure and valve open times. Analysis of the pressure waveforms in the 6 and 24-well modules displayed rise times, peak pressures, and decays with extremely small standard deviations. Data also confirmed that the pressure pulse was distributed evenly throughout the pressure chambers and therefore to each injury well. Importantly, the relationship between substrate deformation and applied pressure was consistent among the multiple wells and displayed a predictable linear behavior in each module.