Focal ischemia due to reduction of cerebral blood flow (CBF), creates 2 zones of damage: the core area, which suffers severe damage, and penumbra area, which surrounds the core and suffers intermediate levels of injury. Objectives: A novel method is introduced, which evaluates mitochondrial function in the core and in the penumbra, during focal cerebral ischemia. Methods: Wistar rats underwent focal cerebral ischemia by middle cerebral artery occlusion (MCAO) for 60 minutes, followed by 60 minutes of reperfusion. Mitochondrial function was assessed by a unique Multi-Site — Multi-Parametric (MSMP) monitoring system, which measures mitochondrial NADH using fluorometric technique, and CBF using Laser Doppler Flowmetry (LDF). Results: At the onset of occlusion, CBF dropped and NADH increased significantly only in the right hemisphere. CBF levels were significantly lower and NADH significantly higher in the core than in the penumbra. After reperfusion, CBF and NADH recovered correspondingly to the intensity of ischemia. Conclusion: Application of the MSMP system can add significant information for the understanding of the cerebral metabolic state under ischemic conditions, with an emphasis on mitochondrial function.