This protocol describes the procedure for analyzing Electron Transport Chain activity of Complexes 1, 2, and 4 in mitochondria isolated from fibroblasts used by the Seifert Lab. It follows the "Electron Transport Chain Protocol" (ETCP) described in: Vekaria, H.J., Kalimon, O.J., Prajapati, P., Velmurugan, G.V., Sullivan, P.G., 2024. An efficient and high-throughput method for the evaluation of mitochondrial dysfunction in frozen brain samples after traumatic brain injury. Front Mol Biosci 11, 1378536. which is a modified version of the "Respirometry in Frozen Samples" (RIFS) assay from: Acin-Perez, R., Benador, I.Y., Petcherski, A., Veliova, M., Benavides, G.A., Lagarrigue, S., Caudal, A., Vergnes, L., Murphy, A.N., Karamanlidis, G., Tian, R., Reue, K., Wanagat, J., Sacks, H., Amati, F., Darley-Usmar, V.M., Liesa, M., Divakaruni, A.S., Stiles, L., Shirihai, O.S., 2020. A novel approach to measure mitochondrial respiration in frozen biological samples. Embo j 39, e104073.