ALP, LDH and protein production are important hepatic functional markers. Hepatocytes proliferation is positively correlated with ALP activity, and synthesis of liver-specific proteins. Hepatocytes release LDH in distress. Current study investigated ALP and LDH activities and total protein concentration in fetal and adult sheep liver tissues and their cultured hepatocytes, as well as the day-wise variations in the ALP activity and total protein concentration in their culture media during their proliferation. Liver tissues were processed by homogenization, and ultrasonication. Processed liver tissues were then subjected to ultracentrifugation. Supernatant was analyzed for ALP activity, LDH activity, and total protein concentration. Hepatocytes were cultured for one week. Their proliferation was characterized by analyzing these parameters in the culture medium on different days. ALP activity was higher in fetal liver tissues. LDH and total protein concentration was higher in adult liver tissues. These parameters increased after culture and were significantly higher in cultured hepatocytes. Day-wise analysis of the culture medium showed that ALP activity reached the maximum values earlier for fetal hepatocytes than that of adult. LDH activity was absent in culture medium, but total protein concentration reached maximum values on seventh day in both the cultures. Present work concluded that the ALP activity increases in the hepatocytes during the cell cuture. Moreover, it is a good marker to investigate hepatocytes proliferation, and fetal hepatocytes have a higher proliferation potential than adult hepatocytes.