Neurons and astrocytes, key elements of the brain active milieu, differ in the metabolic profile and the electron transport chain (ETC) organization. We assessed the redox state of the ETC in two cell types with simultaneous analysis of blood oxygen saturation level (sO2) in awake mice with Raman microspectroscopy. For cell identification, green fluorescent protein (GFP) was expressed in astrocytes, and near-infrared protein (NirFP) was expressed in neurons following AAV injections in the somatosensory cortex (S1). To monitor H2O2, HyPer7 was expressed in the mitochondria of either astrocytes or neurons. Raman spectra recorded from astrocytes and neurons were used to quantify reduced C- and B-type cytochromes. Spectra recorded from blood vessels were used to quantify oxyhemoglobin in the blood. Vessels with sO2 >95% were identified as arterioles, the others as venules. Arterioles dilatated, and sO2 increased in venules during locomotion. The relative amount of reduced cytochromes reversibly increased in astrocytes and decreased in neurons. The latter led to the production of H2O2 in the astrocytic mitochondria but not neuronal. Astrocytic H2O2 generation possibly plays a role in communication between astrocytes and other cells in the brain active milieu.