Background Erlotinib is an orally active antitumor agent. Analyses in vitro using human liver microsomes and recombinant enzymes showed that erlotinib was metabolized primarily by CYP3A4, with a secondary contribution from CYP1A2. Methods A computer-based simulation model, SimCYP(TM), predicted that CYP3A4 contributed to similar to 70% of the metabolic elimination of erlotinib, with CYP1A2 being responsible for the other similar to 30%. A drug-drug interaction study was therefore conducted for erlotinib and a potent CYP3A4 inhibitor, ketoconazole, in healthy male volunteers to evaluate the impact of CYP3A4 inhibition on erlotinib exposure. Results Ketoconazole caused an almost two-fold increase in erlotinib plasma area under the concentration curve and in maximum plasma concentration. This is consistent with the SimCYP(TM) prediction of a two- fold increase in erlotinib AUC, further validating a primary (similar to 70%) role of CYP3A4 in erlotinib elimination. Conclusion Prediction of clinically important drug-drug interaction with SimCYP(TM) using in vitro human metabolism data can be a powerful tool during early clinical development to ensure safe administration of anticancer drugs, which are often co-administered at maximum tolerated doses with other drugs as part of a palliative treatment regimen.