This protocol describes a step-by-step method to isolate functional, purified, and intact mitochondria from three sources: mouse liver, skeletal muscle, and cultured cells. The isolation procedures take approximately 1–2 hours, depending on the tissue source, and the polarographic analysis can be completed in 1 hour. Mitochondria are essential organelles involved in key metabolic reactions and signaling pathways, including apoptosis. The isolation of mitochondria from tissues and cultured cells allows for the study of their function in a controlled environment. The protocol outlines the necessary materials, reagents, and equipment, as well as detailed procedures for isolating mitochondria from different tissues. The isolation process involves homogenization, centrifugation, and washing steps to obtain pure mitochondria. The quality of the isolated mitochondria is assessed using oxygen electrode measurements to evaluate their respiration and coupling. The protocol also includes troubleshooting advice and references to other published protocols for isolating mitochondria from different organs. The goal of the protocol is to obtain high-quality, functional mitochondria that can be used for various biological and pathophysiological studies.This protocol describes a step-by-step method to isolate functional, purified, and intact mitochondria from three sources: mouse liver, skeletal muscle, and cultured cells. The isolation procedures take approximately 1–2 hours, depending on the tissue source, and the polarographic analysis can be completed in 1 hour. Mitochondria are essential organelles involved in key metabolic reactions and signaling pathways, including apoptosis. The isolation of mitochondria from tissues and cultured cells allows for the study of their function in a controlled environment. The protocol outlines the necessary materials, reagents, and equipment, as well as detailed procedures for isolating mitochondria from different tissues. The isolation process involves homogenization, centrifugation, and washing steps to obtain pure mitochondria. The quality of the isolated mitochondria is assessed using oxygen electrode measurements to evaluate their respiration and coupling. The protocol also includes troubleshooting advice and references to other published protocols for isolating mitochondria from different organs. The goal of the protocol is to obtain high-quality, functional mitochondria that can be used for various biological and pathophysiological studies.