Glutathione (GSH) is an important antioxidant that can protect cells under oxidative stress. Thus, a non-invasive method to measure GSH levels in live animals is needed. To map the levels of GSH in mouse brains, a new method using electron paramagnetic resonance (EPR) imaging with nitroxide imaging probes was developed. By analyzing the relationship between reduction rates for nitroxides in brains measured by EPR and brain GSH levels measured by biochemical assay, pixel-based mapping of brain GSH levels was successfully obtained. The newly developed method was applied to a kindling mouse model of epilepsy to clarify the role of GSH.