Assessment of a new chemical entity for cytochrome P450 (CYP) enzyme induction at an early stage in discovery is crucial to prevent potential drug–drug interactions. CYP3A, the most abundant CYP isoform in the liver, metabolizes approximately 50% of drugs currently on the market and is also a highly inducible enzyme. The use of both rat and human hepatocyte culture for the prediction of in vivo CYP3A induction has become refined and validated and is considered a standard in vitro model. The current evaluation of CYP3A enzyme induction involves the use of substrates requiring subsequent analysis of metabolites by high-performance liquid chromatography/mass spectrometry, which adds considerable time and cost. In the present study, we describe the use of a novel luminogenic substrate, luciferin-6′-pentafluoro-benzyl ether (PFBE), which allows for a fast and selective measurement of CYP3A enzyme induction in cultured rat hepatocytes. The extent of induction was evaluated using cells treated for 3 d with the prototypical inducers, dexamethasone, phenobarbital, and pregnenolone 16 alpha-carbonitrile (PCN). Enzyme activity was measured in the treated cells either by the depentafluorobenzylation of luciferin-PFBE or the testosterone 6-β-hydroxylation. Using both methods, dexamethasone and PCN-treated cells exhibited strong CYP3A activity, whereas phenobarbital treatment resulted in a weak response. The fold induction varied between both methods, but this variability can be controlled by normalizing data from each treatment to a positive control. The results indicate that luciferin-PFBE is an attractive alternative to the use of conventional substrate, testosterone, providing a sensitive, robust, and rapid method compatible with the multiwell plate format for the assessment of CYP3A induction.