The liver plays an important role in the clearance of endogenous and exogenous compounds, including drugs. As hepatic uptake is the first step in hepatic clearance, any change in the former process directly affects the overall intrinsic hepatic clearance. Several uptake transporters are expressed on the basolateral membranes of hepatocytes and mediate the hepatic uptake of hydrophilic charged compounds that cannot easily penetrate the plasma membrane. As the substrate specificities of these individual drug transporters are broad and overlap, compounds are often recognized by multiple uptake transporters. Thus, knowledge of the contribution that each transporter makes to the hepatic uptake of a compound is important for predicting the extent to which hepatic uptake clearance will change if the activity of a specific transporter is altered by a genetic polymorphism or a drug-drug interaction. Human cryopreserved hepatocytes are now commercially available and can be used for studying hepatic uptake clearance. In this chapter, we describe a method for using isolated hepatocytes to estimate the in vivo uptake clearance of compounds and the quantitative contribution of each uptake transporter to the overall hepatic uptake of anionic compounds.