Mitochondria are intracellular organelles where most fundamental processes of energy transformation within the cell are located. They also play important roles in programmed cell death (apoptosis), free radical formation, and signal transduction. In addition, mitochondria host genes encoding several important proteins. Studying isolated mitochondria is therefore crucial for better understanding cell functioning. The present article describes a relatively simple and handy procedure for isolation of crude mitochondrial fraction from cultivated mammalian and human cells. It consists of mechanical homogenization and fractionating centrifugation. Assays of checking mitochondrial competence by measuring membrane potential formation and coupled respiration are also presented.