Abstract <p>The aim of the present study was to develop a sensitive, high-throughput 96-well plate ultraviolet spectrometric method for the determination of peroxide value at trace levels in reagents and packaging materials. The analysis was carried out under acid-free conditions to minimize or prevent reactions between iodine molecules and oxygen in the air, thereby ensuring the accuracy and repeatability of the experiments. The wavelength was set at 360 nm. The analytical procedure was validated by evaluating linearity (<i>r</i>² = 0.9998), precision (intra- and inter-day precision were less than 0.90 and 6.20%, respectively.), accuracy (100–110% of the spiked amount), and limits of detection (<b>LOD</b>) and quantification (<b>LOQ</b>). The LOD and LOQ were confirmed to be 20 and 50 ng/mL, respectively. This high-throughput technique reduces the risk of chemical changes during analysis due to time inconsistencies, which benefits the accuracy and reproducibility of results. The proposed method was then successfully applied to determine trace amounts of peroxides in ethyl acetate and packaging material samples. Overall, the present study offers an accurate and sensitive method for measuring trace-level peroxides.</p>