Radiation resistance is a significant issue, leading to the failure of radiation therapy and tumor relapse. This study aims to select an X-ray-resistant S. cerevisiae strain to evaluate deregulated proteins in response to high ionizing radiation exposure, with the goal of identifying candidate molecular biomarkers. A wild-type (wt) strain was exposed to increasing doses of X-rays until a resistant strain was obtained. Survival was assessed, and proteomic analysis was performed via mass spectrometry. The resistant cells exhibited 9.38 times greater resistance than the wt strain. Several deregulated proteins were identified. These findings indicate that acquired radiation resistance at high doses is associated with the increased expression of GAPDH and ENO1, which are proposed as candidate molecular biomarkers associated with acquired radiation tolerance, pending further functional validation. Data are available via ProteomeXchange with identifier PXD074114.