Isolated perfused liver and cultures of rat hepatocytes were assessed for the quantitative evaluation of hepatotoxicity. Release of de novo biosynthesized plasma proteins and acid hydrolases into perfusion or culture media was taken as an indication of the integrity of hepatocytes in both systems. The activities of six acid hydrolases, α‐L‐fucosidase, α‐D‐galactosidase, β‐D‐galactosidase, β‐D‐N‐acetylgalactosaminidase, β‐D‐N‐acetylglucos‐aminidase, and cathepsin D, were assayed in collagenase‐segregated hepatocytes and in monolayer cultures of rat liver cells obtained via collagenase perfusion of rat liver. In situ, liver perfusion with collagenase led to a loss of 45 ± 5% of the total acid hydrolase activity in the mitochondrial‐lysosomal pellet of the liver cells with concomitant increase of these enzymes in the cytosol. In monolayer cultures over a period of 30 h, increased activity of cathepsin D, β‐D‐galactosidase, and β‐D‐N‐acetylglucosaminidase in the mitochondrial‐lysosomal pellet and the cytosol fraction was evident with concurrent biosynthesis of plasma proteins. The use of radioactive tracing techniques with the isolated perfused liver revealed that the rate of catabolism of intracellular protein was ∼5 times that of plasma protein synthesis. Both methods described here are suitable for the study of the effects of toxins on the function of hepatocytes.