Purpose: To investigate whether a plasmid DNA-based detector can be used to quantify the relative biological effectiveness (RBE) of a superficial 100 kVp beam (test radiation) using a 6 MV beam as the standard radiation. Methods: Recent developments in clinical radiotherapy modalities including heavy particle, FLASH, and MRI-guided radiotherapy would benefit from a radiobiological-based detector that can assess RBE with respect to DNA damage. To this end, DNA detectors were constructed by pipetting plasmid DNA, pBR322 (Thermo Fisher Scientific, Inc.), into nine 6.28 mm3 cylindrical wells drilled into biocompatible near tissue equivalent material (polycarbonate). Each well contained 3 ul of DNA solution with a concentration of 18 ng/ul for a total of 27 ul per detector. Each detector was sealed using aluminum foil seals. Five DNA-based detectors were sent from San Diego State University (San Diego, CA) to the Odette Cancer Centre – Sunnybrook Hospital (Toronto, Canada), two controls and two to be irradiated to 20 Gy using a 100 kVp x-ray beam (Xstrahl 300) and a 6 MV x-ray beam (Elekta Synergy Linear Accelerator). Agarose gel electrophoresis was used to separate the plasmid DNA open circular, linear, and supercoiled isoform bands corresponding to single strand breaks (SSBs), double strand breaks (DSBs), and no strand breaks, respectively. The gel band intensities were imaged to quantify the relative yields of SSBs and DSBs. The resulting band yields were modeled to find the conformational yields as a function of dose and assess the RBE differences between the test and standard radiation beams. Results: At 20 Gy irradiation, the 100 kVp irradiated DNA exhibited 1.7 േ 1.4% more SSBs than the DNA irradiated with 6 MV, as shown to be significant by the t-test (p < 3.7 ൈ 10ିସ). Furthermore, a SSB yield endpoint of 20% for the 100 kVp energy gave an RBE of 1.14 േ 0.14. Conclusion: A statistically significant difference was observed in the production of SSBs when plasmid DNA was irradiated by the 100 kVp and 6 MV beams. Although an SSB RBE > 1 was observed for the 100 kVp beam, no significant difference in DSBs was found in the DNA between the two beam qualities.