Avoiding drug-drug interactions (DDIs) mediated through inhibition of cytochrome P450 (CYP) activity is highly desirable. Direct inhibition (DI) of CYP through new chemical entities (NCEs) or time-dependent inhibition (TDI) through reactive metabolites should be elucidated at an early stage of drug discovery research. In particular, TDI of CYP occurring through reactive metabolites may be irreversible and even sustained, causing far more serious DDIs for TDIs than for DIs. Furthermore, it is important to ascertain whether an NCE inhibits multiple CYP isoforms. Hence, using a cocktail-substrate approach that we previously established (in which the activity of 8 CYP isoforms is simultaneously evaluated in a single run), we evaluated the IC50 values of direct inhibitors and TDI parameters (kobs, shifted IC50, KI and kinact) of time-dependent inhibitors that affect multiple CYP isoforms. The IC50 values for 8 CYP isoforms obtained using the cocktail-substrate approach were nearly identical to values previously reported. The TDI parameters for CYP1A2, 2C9, 2C19, 2D6, and CYP3A4/5 obtained using the cocktail-substrate approach were also nearly identical to those obtained using a single-substrate approach. Thus, the cocktail-substrate approach is useful for evaluating DI and TDI in the early stages of drug discovery and development processes.