The increasing incidence of cancer emphasizes the urgent need for the development of novel and more effective therapeutic strategies. Glioblastoma multiforme remains one of the most aggressive and treatment-resistant tumors with a dismal prognosis. In this study, 12 nm cubic palladium nanoparticles (Pd NCs) were synthesized as a cost-effective alternative to gold nanoparticles (Au NPs) for use as nano-radiosensitizers in high-energy proton beam therapy. To further improve their biocompatibility and stability in biological environments, Pd NCs were covalently functionalized with thiolated poly(ethylene glycol) (PEG-SH). In vitro assays (MTS test and clonogenic assay) on LN229 and U118 glioblastoma cell lines demonstrated that PEGylation significantly reduced cytotoxicity. For LN229 cells, Pd NCs and PEGylated Pd NCs (Pd NCs-PEG) exhibited comparable radiosensitizing effects at equivalent concentrations, whereas in U118 cells, Pd NC-PEG induced a significantly stronger effect. Holotomographic microscopy confirmed larger NP uptake in U118 compared to LN229 cells, suggesting cell-line-dependent internalization efficiency. Finally, Fouriertransform infrared spectroscopy (FTIR) revealed NP- and/or proton beam-induced biochemical changes in glioblastoma cells, particularly in protein and carbohydrate content. These findings indicate that Pd NCs possess promising radiosensitizing properties and that PEGylation enhances their biocompatibility, offering a potential strategy to improve the overall efficacy of proton therapy outcomes for glioblastoma.