As a highly efficient delivery system, lentiviral vectors (LVs) have become a powerful tool to assess the antiviral efficacy of RNA drugs such as short hairpin RNA (shRNA) and decoys. Furthermore, recent advanced systems allow controlled expression of the effector RNA via coexpression of a tetracycline/doxycycline (DOX) responsive repressor (tTR-KRAB). Herein, this system was utilized to assess the antiviral effects of LV-encoded shRNAs targeting three conserved regions on the pregenomic RNA of hepatitis B virus (HBV), namely the region coding for the reverse transcriptase (RT) domain of the viral polymerase (LV-HBV-shRNA1), the core promoter (CP; LV-HBV-shRNA2), and the direct repeat 1 (DR1; LV-HBV-shRNA3). Transduction of just the LV-HBV-shRNA vectors into the stably HBV expressing HepG2.2.15 cell line showed significant reductions in secreted HBsAg and HBeAg, intracellular HBcAg as well as HBV RNA and DNA replicative intermediates for all vectors, however, most pronouncedly for the DR1-targeting shRNA3. The corresponding vector was therefore applied in the DOX-controlled system. Notably, strong interference with HBV replication was found in the presence of the inducer DOX whereas the antiviral effect was essentially ablated in its absence; hence, the silencing effect of the shRNA and consequently HBV replication could be strictly regulated by DOX. This newly established system may therefore provide a valuable platform to study the antiviral efficacy of RNA drugs against HBV in a regulated manner, and even be applicable in vivo.