Accurate prediction of human pharmacokinetics (PK) is important for the choice of promising compounds in humans. As the predictability of human PK by an empirical approach is low for drugs with species-specific PK, the utility of a physiologically based pharmacokinetic (PBPK) model was verified using 16 reference drugs hepatically metabolized. After the prediction method for total clearance (CLtot) and distribution volume at steady state (Vdss) in the conventional PBPK model had been optimized, plasma concentrations following a single oral administration of each reference drug to healthy volunteers were simulated and the prediction accuracy for human PK was compared between empirical approaches and the optimized PBPK model. In the drugs with low species-specific CLtot, there was little difference in predictability for maximum concentration (Cmax), time to maximum plasma concentration (Tmax) and area under the curve (AUC) (AAFE: 1.3 to 2.4). In contrast, the optimized PBPK model predicted Cmax and AUC of the drugs with high species-specific CLtot with lower AAFE (Cmax and AUC: 2.8 and 3.2, respectively) than those of the empirical approach (Cmax and AUC: 2.6 to 4.9 and 3.9 to 10.7, respectively). Therefore, the optimized PBPK model is useful for human PK prediction of drugs with species-specific CLtot.