Abstract: Background Approximately 50% of newly diagnosed cancer patients worldwide receive radiotherapy, most commonly with X-rays. Proton therapy has emerged as a promising alternative, offering complementary advantages over conventional X-rays due to its highly localized energy deposition at the Bragg peak, thereby reducing radiation exposure to surrounding healthy tissues. In recent years, advances in both technology and biomedicine have driven significant progress in proton therapy, with ongoing research aiming to minimize toxicity and long-term side effects while improving patients' quality of life. An emerging research direction investigates the combination of proton therapy with irradiated nanoparticles acting as radiosensitizers. This approach seeks synergistic effects that could further improve therapeutic outcomes. These effects arise from a complex interplay of physical, chemical, and biological mechanisms. Although previous in vitro and in vivo studies report promising radiosensitization effects of nanoparticles, the literature also includes contradictory results, and the underlying mechanisms remain under debate. This highlighting the need for systematic studies under well-controlled irradiation conditions of different nanoparticles and beam parameters, which are often difficult to perform due to the limited availability of particle accelerators and facilities suitably adapted for such experiments.Purpose This work investigates the radiosensitizing effect of gold nanoparticles under proton irradiation and optimizes the irradiation setup to enable more efficient and reproducible future experiments.Methods HeLa cells were used as the biological model for the radiobiological experiments. Irradiation conditions were optimized by incorporating a Mylar film into the culture plates to maintain a minimal medium volume and ensure rapid post-irradiation coverage. Cells with and without AuNPs were irradiated with 12.6 MeV proton beams. Radiosensitivity was assessed using clonogenic survival assays, and the radiosensitizing effect, together with its associated uncertainty, was quantified. DNA damage analysis was also performed to corroborate the observed effects and explore underlying mechanisms.Results The radiobiology experiments confirmed and quantified a significant radiosensitizing effect of gold nanoparticles (AuNPs) on HeLa cells at doses from 4 Gy, with Sensitization Enhancement Ratio (SER) values of 1.53 +/- 0.07 in 2022 and 1.30 +/- 0.20 in 2023 using 50 nm diameter AuNPs. Experiments in 2023 with 20 nm diameter AuNPs yielded a comparable SER of 1.38 +/- 0.18, indicating no significant size-dependent differences. The radiosensitizing effect exhibited dose-dependent amplification, with higher doses producing greater cell killing. Linear-Quadratic (LQ) model fits showed an increased alpha$\alpha$ parameter in the presence of AuNPs, consistent with an increase in non-repairable damage. This trend was further supported by double-strand break assays. Furthermore, oxidative stress assays showed that AuNPs do not increase basal ROS levels, but enhance ROS production under oxidative stress conditions.Conclusion The radiobiology experiments presented quantitatively confirm that both 20 and 50 nm diameter AuNPs enhance the radiosensitivity of HeLa cells under proton irradiation. The radiosensitizing effect increases with dose, indicating a dose-dependent response, while no significant differences were observed between nanoparticle sizes. Consistent trends of increased DNA double-strand breaks further support the observed radiosensitization. Evidence from oxidative stress assays suggests that AuNPs may enhance ROS production under oxidative conditions, potentially contributing to the observed radiosensitization and DNA damage.