The hepatitis A virus (HAV) infection causes acute hepatitis. HAV also induces acute liver failure or acute-on-chronic liver failure; however, no potent anti-HAV drugs are currently available in clinical situations. For anti-HAV drug screening, more convenient and useful models that mimic HAV replication are needed. In the present study, we established HuhT7-HAV/Luc cells, which are HuhT7 cells stably expressing the HAV HM175-18f genotype IB subgenomic replicon RNA harboring the firefly luciferase gene. This system was made by using a PiggyBac-based gene transfer system that introduces nonviral transposon DNA into mammalian cells. Then, we investigated whether 1134 US Food and Drug Administration (FDA)-approved drugs exhibited in vitro anti-HAV activity. We further demonstrated that treatment with tyrosine kinase inhibitor masitinib significantly reduced both HAV HM175-18f genotype IB replication and HAV HA11-1299 genotype IIIA replication. Masitinib also significantly inhibited HAV HM175 internal ribosomal entry-site (IRES) activity. In conclusion, HuhT7-HAV/Luc cells are adequate for anti-HAV drug screening, and masitinib may be useful for the treatment of severe HAV infection.