Inhibition of the bile salt export pump (BSEP) has been recognized as a key factor for the development of drug-induced cholestasis (DIC). The risk of DIC in human was previously assessed using in vitro BSEP inhibition data (IC50) and unbound systemic drug exposure data under assumption of the “free-drug hypothesis”. This concept, however, is unlikely valid for organs such as the liver, where unbound intracellular drug concentrations are affected by active transport and metabolism. To investigate this hypothesis we experimentally determined the liver-to-blood partition coefficients (Kpuu) for 18 drug compounds following the theory of the hepatic Extended Clearance Model (ECM). Unbound intrahepatic concentrations were calculated from clinical exposure (systemic and hepatic inlet) and measured Kpuu data. Consequently, corresponding safety margins against BSEP IC50 values were determined and compared to the clinical incidence of DIC. Depending on the ECM class of a drug, Kpuu values deviated up to 14-fold from unity and unbound intrahepatic concentrations were affected accordingly. The use of Kpuu-based safety margins allowed to separate clinical cholestasis frequency classes (no cholestasis, cholestasis in ≤ 2% or in > 2% of subjects) for almost the entire set of compounds. This assessment was found to be superior as compared to using unbound extracellular concentrations as surrogate matrix. Furthermore, the assessment of Kpuu according to ECM provides useful guidance for quantitative evaluation of genetic and physiological risk factors for the development of cholestasis.