Primary human hepatocytes are necessary to evaluate cytotoxicity, drug metabolism, and drug-drug interactions for candidate compounds in early-phase drug discovery and development. However, these analyses are often hampered by limited resources and functional or genetic variation among lots. HepaRG human hepatocellular carcinoma cells can differentiate into mature hepatocyte-like cells (HepLCs) that possess similar metabolic activity to human hepatocytes. We previously established transgenic HepaRG cells carrying a dual reporter that express red fluorescent protein (RFP) under the transcriptional regulation of CYP3A7 in the hepatoblast-like cell state and enhanced green fluorescent protein (EGFP) under the transcriptional regulation of CYP3A4 following HepLC differentiation. In this study, we successfully isolated a subclone of transgenic CYP3A4G/7R HepaRG cells with an improved HepLC differentiation potency. Midazolam metabolism by CYP3A4 in these HepLCs was comparable to that in wild-type HepLCs. The EGFP fluorescence intensity was greatly induced by rifampicin (RIF) treatment. There was a strong correlation between fluorometric and metabolic analyses. The fold change in EGFP-positive cells was comparable to those in the CYP3A4 mRNA level and luminescence of proluciferin metabolites. RIF treatment and cell proliferation increased the RFP-positive cell number. Thus, CYP3A4G/7R HepLCs provide a real-time, multiwell-based system to co-evaluate CYP3A4 induction and hepatic regeneration.