Assessment of time-dependent inhibition (TDI), especially CYP3A4, is an important parameter for preclinical and clinical development. The use of human liver microsomes (HLM) is the most common in vitro matrix to assess TDI, but this often leads to an overprediction of an actual effect observed clinically. Recently, the use of human hepatocytes has been hypothesized as a more relevant and possibly predictive matrix for the assessment of CYP3A4 TDI. Our work evaluates and optimizes three different human hepatocyte assays for the assessment of CYP3A4 TDI using pooled cryopreserved human hepatocytes. Using two of the optimized methods, the time-dependent inhibition kinetic parameters (KI and kinact) for four known CYP3A4 TDI (diltiazem, erythromycin, verapamil, and troleandomycin) were determined. When comparing TDI in HLM, the KI values from hepatocytes were in general 4- to 13-fold higher than that in HLM, whereas the kinact values in human hepatocytes were similar or slightly higher or lower depending on the inhibitor. The inactivation potency (kinact/KI) for four tested CYP3A4 inactivators in human hepatocytes was generally lower than that in HLM due to either lower affinity (KI) or lower inactivation rate (kinact) or both. When drug interactions were simulated with Simcyp® using either HLM or human hepatocyte data, the predictions using the kinetic parameters from human hepatocytes resulted in a much better simulated change in pharmacokinetics compared with observed clinical data.