We have proposed a new method which can be applied in assessing the intracellular production of hydrogen peroxide. Using this assay we have examined the hydrogen peroxide generation during the L-glutamate induced oxidative stress in the HT22 hippocampal cells. The detection of hydrogen peroxide is based on two crucial reagents cis-[Cr(C2O4)(pm)(OH2)2]+ (pm denotes pyridoxamine) and 2-ketobutyrate. The results obtained indicate that the presented method can be a promising tool to detect hydrogen peroxide in biological samples, particularly in cellular experimental models.