The availability of a reliable in vitro assay to evaluate time-dependent inhibition (TDI) of cytchrome P450 enzymes by novel compounds is essential for the identification of candidate medicines. We have evaluated three assay methods, making use of 59 marketed compounds and 28 novel GSK compounds. Recombinant bactosomes expressing the CYP3A4 isozyme were used with two fluorescence-based methods: a “Re-addition” assay and a “30 min” assay. The third method evaluated used pooled human liver microsomes (PHLM) with LC-MS/MS detection (the data for GSK compounds were evaluated in this study, whereas data for marketed drugs were reported recently). Our evaluation showed that the Re-addition method is comparable to the LC-MS/MS method in terms of predictivity and reproducibility. In conclusion, Re-addition method is inexpensive, and provides a simple assessment of the risk of TDI for novel compounds. This assay is particularly appropriate for use during the early stages of drug discovery.