Proton therapy offers superior dose localization, yet the biological effects of low-energy protons relevant to superficial tissues remain underexplored. We report the design and validation of a proton irradiation setup developed at the Tandem Accelerator of NCSR “Demokritos” for controlled radiobiological experiments. Monte Carlo simulations using Geant4 and Monte Carlo Damage Simulation (MCDS—Monte Carlo Damage Simulation) were used to determine proton energy spectra, linear energy transfer (LET), and predicted DNA damage yields. A single layer (15–20 μm in thickness) of human keratinocytes (HaCaT) was irradiated at doses from 0.65 to 3.65 Gy, and γ-H2AX foci were quantified as markers of tracks including one or more DNA double-strand breaks. The system achieved a uniform dose rate of 0.37 Gy/min, as calculated with Geant4, with a mean proton energy of 4.1 MeV (LET ≈ 8 keV/μm). A strong correlation (R² = 0.93) was observed between proton dose and γH2AX foci per nucleus (~10 foci/Gy), reflecting damage-inducing proton tracks rather than individual DNA double-strand breaks. At higher doses, an increased fraction of cells exhibited pan-nuclear γH2AX staining, characterized by a diffuse γH2AX signal throughout the nucleus and commonly associated with extensive or clustered DNA damage and global chromatin phosphorylation. These responses are consistent with the well-established dense ionization patterns produced by low-energy protons, as indicated by the LET spectrum and supported by MCDS-predicted clustered damage yields. While the γH2AX assay does not directly resolve simple versus complex DNA lesions, the agreement between Monte Carlo modeling and the observed cellular stress responses indicates that the irradiation platform reliably reproduces the expected biological signatures of low-energy proton exposure. Consequently, the developed system provides a robust experimental tool for systematic investigations of cellular radiosensitivity and radiotoxicity, with potential applications in skin dosimetry and radioprotection.