Human retinal pigment epithelium cells under hydrogen peroxide-induced oxidative stress and a ligustrazine-based protective effect were investigated using laser tweezers Raman spectroscopy. Protein and lipid were significantly affected by oxidative damage, along with increased reactive oxygen species (ROS) level within cells. The effects of ligustrazine against the reaction of ROS with protein seemed to be able to inhibit such damages but were limited during the desamidization of amides, along with additional effect on nucleic acid base and DNA phosphoric acid skeleton. This work laid the basis for both understanding the molecular mechanisms of oxidative stress-induced injury and highlighting possible biomarkers in retinal diseases.