Diabetes has a systemic effect on the metabolism and can be linked to higher risks of severe medical conditions, such as ischemic stroke. Hyperglycemia leads to more extensive cerebral damage during stroke and, as a result, to more severe consequences. One of the main damaging factors of the stroke is the reactive oxygen species generation occurring in mitochondrial respiratory chain (electron transport chain, ETC). We used in vivo Raman microspectroscopy with the chronic cranial windows implantation to assess the redox state of mitochondria ETC in neurons of awake mice under normal and hyperglycemic conditions. We then estimated the levels of H2O2 production in neuronal mitochondria in vitro and in vivo using HyPer7 biosensor and a fiber-optic interface technology. We demonstrated that high blood glucose levels led to the increase in the relative amount of reduced cytochromes in the mitochondrial respiratory chain in neurons of awake mice indicating loading of the ETC with electrons and the increase in oxygen superoxide generation. However, hyperglycemia did not affect the H2O2 production in the mitochondria of neurons in the ischemic core, while significantly exacerbating the consequences of the stroke pathogenesis. It is suggested, that hyperglycemia leads to the mitochondria-related production of the reactive oxygen species, but not H2O2.