Humans can be exposed to ionizing radiation, due to various reasons, whose structural effects on biological membranes are not well defined. The current study aims to understand the ionizing radiation-induced structural and functional alterations in biomolecules of brain membranes using Fourier transform infrared (FT-IR) spectroscopy using rat animal models. For this purpose, 1000 cGy of ionizing radiation was specifically directed to the head of Sprague Dawley rats. The rats were decapitated after 24 h. The results revealed that the lipid-to-protein ratio decreased and that irradiation caused lipid peroxidation and increases in the amounts of olefinic =CH, carbonyl, and methylene groups of lipids. In addition, ionizing radiation induced a decrease in membrane fluidity, disordering of membrane lipids, strengthening of the hydrogen bonding of the phosphate groups of lipid head-groups, and weakening in the hydrogen bonding of the interfacial carbonyl groups of lipids. Radiation further caused significant decrements in the α-helix and turns, and significant increments in the β-sheet and random coil contents in the protein structure. Hierarchical cluster analyses, performed in the whole region (3030-1000 cm(-1)), lipid (3030-2800 cm(-1)), and protein (1700-1600 cm(-1)) regions separately, successfully differentiated the control and irradiated groups of rat brain membranes and showed that proteins in the membranes are affected more than lipids from the damages induced with ionizing radiation. As a result, the current study showed that FT-IR spectroscopy can be used successfully as a novel method to monitor radiation-induced alterations on biological membranes.