Glutathione (GSH) concentrations in cells are vital indicators of cell health; therefore quantification of GSH is imperative for the diagnosis of diseases at an early stage. Rhodamine 6G capped gold nanoparticle (R6G@AuNPs) is developed as a probe for detecting free radical (•OH) induced oxidation of glutathione (GSH). The interaction between AuNPs and R6G molecules leads to the quenching of the intense fluorescence of R6G at 550 nm. In absence of •OH radicals, the GSH present in the sample efficiently displaces the R6G on the surface of AuNPs leading to increased fluorescence intensity in the supernatant. •OH radical (generated by radiolysis) leads to the oxidation of GSH to GSSG, which cannot displace R6G and therefore the fluorescence intensity at 550 nm is diminished with increasing dose. Systematic optimization of concentration of R6G, GSH and irradiation dose will help in the development of a low cost, sensitive method for the detection of •OH induced oxidation of GSH.