Proteins are the major target for reactive oxygen species as a result of their abundance in biological systems. Stress conditions induce the upsurge in the level of reactive oxygen species causing protein oxidation recognized by the formation of protein radicals, fragments, aggregates, and subsequent degradation. In the current study, we used the immuno-spin trapping technique for the detection of protein radicals in vivo and in vitro. We performed western blotting for the characterization of proteins involved in the formation of protein radicals, fragments, and aggregates. We observed that light stress triggered the upsurge in the level of hydroxyl radical and singlet oxygen. Hydroxyl radical and singlet oxygen induced protein oxidation, which is recognized by the formation of reactive carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) protein radicals. Generation and kinetic behavior of reactive oxygen species and molecular oxygen in spinach leaves have been studied under abiotic stress. We used electrochemical biosensor techniques for the real-time monitoring of superoxide anion radical and hydrogen peroxide formation and two-dimensional imaging of light-induced oxygen evolution. Immuno-spin trapping technique for the detection and characterization of protein radical formation is imperative for better understating of the mechanism of oxidative modification in PSII proteins under high light stress and electrochemical biosensor techniques can offer precise information on the qualitative determination of superoxide anion radical, hydrogen peroxide and molecular oxygen.Proteins are the major target for reactive oxygen species as a result of their abundance in biological systems. Stress conditions induce the upsurge in the level of reactive oxygen species causing protein oxidation recognized by the formation of protein radicals, fragments, aggregates, and subsequent degradation. In the current study, we used the immuno-spin trapping technique for the detection of protein radicals in vivo and in vitro. We performed western blotting for the characterization of proteins involved in the formation of protein radicals, fragments, and aggregates. We observed that light stress triggered the upsurge in the level of hydroxyl radical and singlet oxygen. Hydroxyl radical and singlet oxygen induced protein oxidation, which is recognized by the formation of reactive carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) protein radicals. Generation and kinetic behavior of reactive oxygen species and molecular oxygen in spinach leaves have been studied under abiotic stress. We used electrochemical biosensor techniques for the real-time monitoring of superoxide anion radical and hydrogen peroxide formation and two-dimensional imaging of light-induced oxygen evolution. Immuno-spin trapping technique for the detection and characterization of protein radical formation is imperative for better understating of the mechanism of oxidative modification in PSII proteins under high light stress and electrochemical biosensor techniques can offer precise information on the qualitative determination of superoxide anion radical, hydrogen peroxide and molecular oxygen.