This protocol describes a step-by-step method for isolating functional mitochondria from mouse liver, muscle, and cultured fibroblasts. The isolation process, which takes 1-2 hours depending on the source, involves several critical steps such as cell or tissue homogenization, centrifugation, and resuspension in isolation buffers. The quality of the isolated mitochondria is assessed using polarographic analysis to measure their respiration. The protocol emphasizes the importance of maintaining mitochondrial integrity and purity, with specific instructions for avoiding contamination and preserving functionality. The authors also provide troubleshooting tips and a detailed timeline for each step, ensuring that researchers can effectively isolate and use high-quality mitochondria for various biological and pathological studies.This protocol describes a step-by-step method for isolating functional mitochondria from mouse liver, muscle, and cultured fibroblasts. The isolation process, which takes 1-2 hours depending on the source, involves several critical steps such as cell or tissue homogenization, centrifugation, and resuspension in isolation buffers. The quality of the isolated mitochondria is assessed using polarographic analysis to measure their respiration. The protocol emphasizes the importance of maintaining mitochondrial integrity and purity, with specific instructions for avoiding contamination and preserving functionality. The authors also provide troubleshooting tips and a detailed timeline for each step, ensuring that researchers can effectively isolate and use high-quality mitochondria for various biological and pathological studies.