The fraction of an absorbed drug metabolized by the different hepatic CYP enzymes, relative to total hepatic CYP metabolism (fm,CYP), can be estimated by measuring the inhibitory effects of presumably selective CYP inhibitors on the intrinsic metabolic clearance of a drug using human liver microsomes (HLM). However, the chemical inhibition data are often affected by cross-reactivities of the chemical inhibitors used in this assay. To overcome this drawback, the cross-reactivities exhibited by six chemical inhibitors (furafylline, montelukast, sulfaphenazole, ticlopidine, quinidine and ketoconazole) were quantified using specific CYP enzyme marker reactions. The determined cross-reactivities were used to correct the in vitro fm,CYPs of nine marketed drugs. The corrected values were compared with reference data obtained by PBPK simulation using the software SimCYP. Uncorrected in vitro fm,CYPs of the nine drugs showed poor linear correlation with their reference data (R2 = 0.443). Correction by factoring in inhibitor cross-reactivities significantly improved the correlation (R2 = 0.736). Hence, correcting in vitro chemical inhibition results for cross-reactivities appears to offer a straightforward and easily adoptable approach to provide improved fm,CYP data for a drug.