The liver is composed of hepatocytes, endothelial, Kupffer, fat-storing, and pit cells. This chapter describes a technique for isolating hepatocytes, which represent 60–65% of the cells of the liver and about 80% of the volume of the organ (since they are larger than the other cells) (1–3). Two years after Howard et. al. (4) discovered that collagenase was a very important tool in the separation of liver cells, Berry and Friend (5) introduced the technique of collagenase perfusion of the liver.