The liver is a unique organ that is endowed with a plethora of specialized functions. Most of its functional traits are controlled by hepatocytes. Primary hepatocytes have been used widely in in vitro models to understand the biological processes occurring in the liver. There are a number of methods used to separate hepatocytes, but the cell activity and purity are much lower in this condition. On the basis of previous research, in this study, the two-step collagenase perfusion technique was used for isolating hepatocytes. The key proteins of hepatocytes, cytokeratin-18 (CK-18) and albumin (ALB), were used to identify cells, and their contents were evaluated by immunohistochemistry and Western blotting. The results showed that the isolated hepatocytes comprised more than 96% of the corresponding protein volume stability. Therefore, this method was demonstrated to be reliable for identifying hepatocytes.