Stroke is the second leading cause of mortality worldwide, with retinal ischemia as its prominent complication. However, the pathology of retinal ischemia has not been fully elucidated, resulting in a lack of effective treatment. Stem cell therapy has been suggested to be therapeutic in retinal ischemia, with mitochondrial transfer potentially one of the underlying mechanisms. To investigate the mitochondrial function in retinal ischemia and the potential of mitochondrial transfer from mesenchymal stem cells (MSCs), in vivo middle cerebral artery occlusion (MCAO) model and in vitro oxygen-glucose deprivation (OGD) model were utilized in combination. In vivo, rats subjected to MCAO were randomly administered intravenous MSCs or vehicles. Laser doppler was used to measure the blood flow in the brain and the eye, along with immunohistochemical staining for assessing cellular degeneration. In vitro, retinal pigment epithelium (RPE) cells exposed to OGD were cocultured with or without MSCs. Mitochondrial function was measured by mitochondrial respiration, mitochondrial network analysis, mitochondria live cell imaging, and immunocytochemistry. The results demonstrated improved cell survival and restored mitochondrial function following MSC therapy. This chapter details the protocols necessary to produce the in vivo and in vitro models of ischemic stroke along with an assessment of mitochondrial function. Elucidating the mechanisms of mitochondrial transfer will further the knowledge in regenerative medicine and may enable new targets of therapeutics for stroke, especially for retinal ischemia.