Reactive oxygen species (ROS) are key mediators in a wide range of physiological and pathological processes, including aging and the development of chronic diseases. While excessive ROS levels can damage proteins, lipids, and DNA, controlled concentrations are essential for normal cellular signaling and homeostasis. This dual role of ROS has created a growing need for sensitive, reproducible, and cost-effective methods to monitor oxidative stress. In this study, we developed and optimized a spectrophotometric method for the quantification of carbonylated proteins (CP), which are established markers of oxidative protein damage. The method is based on the reaction of CP with 2,4-dinitrophenylhydrazine (2,4-DNPH), followed by absorbance measurement in a 96-well microplate format. This microplate-based approach enables simultaneous processing of a large number of samples, reduces reagent and sample consumption, and significantly increases throughput and efficiency. The optimized protocol demonstrated enhanced analytical performance, reduced cost per analysis, and shorter execution time compared to conventional assays. These characteristics make it well suited for both clinical diagnostics and basic research applications. Moreover, the method can be used to monitor oxidative stress in various pathological conditions and to assess the effectiveness of antioxidant therapies. Overall, the proposed approach offers a practical and scalable solution for studying protein oxidation and redox-related cellular dysfunctions.