We investigated the effect of proton FLASH radiation on plasmid DNA. Purified supercoiled pBR322 plasmids were irradiated with clinical doses (≤10 Gy) of protons at ultra-high and conventional dose rates using the Paul Scherrer Institute (PSI) isochronous cyclotron. The proton beam in this clinical facility has been validated to produce the FLASH effect in preclinical models. Plasmid samples were irradiated under various oxygen tensions, scavenger levels, pH conditions and Fe (II) concentrations as these biochemical parameters vary across tissues and tumors. Over the range of doses used, plasmid DNA strand breaks were found to be dose rate independent at all conditions investigated. Irradiation within the Bragg peak and spread-out Bragg peak increased clustered strand breaks, except in the presence of scavengers. With this model system, we demonstrate conclusively that plasmid DNA strand breakage is dose rate independent at doses below 10 Gy and does not constitute a high throughput assay endpoint predictive of the biological effect of FLASH.