Nowadays, non-communicable (chronic) diseases are considered responsible for the majority of global deaths. Moreover, cancer is predicted to be the leading cause of human death in the 21st century. One of the most common treatment of cancer diseases is radiotherapy, which applies ionizing radiation (e.g. X-rays or protons) to damage vital biomolecules (mainly DNA) and induce cell death. Recently, much attention has been focused on the application of vibrational spectroscopy methods for the detection and analysis of radiation-induced damage and response of cancer cells. Therefore, I will present the use of Raman and AFM-IR imaging techniques to monitor subcellular changes induced by ionizing radiation (X-ray) in PC-3 prostate cancer cells.