In T cell-based cancer immunotherapy, tumor antigen (Ag)-specific CD8 cytotoxic T lymphocytes (CTLs) can specifically target tumor Ags on malignant cells. This promising approach drove us to adopt this strategy of T cell transfer (ACT)-based immunotherapy for chronic viral infections. Here, we describe the generation of hepatitis B virus (HBV) Ag-specific CTLs from induced pluripotent stem cells (iPSCs), i.e., iPSC-CTLs. Ag-specific iPSC-CTLs can target HBV Ag cells and infiltrate into the liver to suppress HBV replication in a murine model. For complete details on the use and execution of this protocol, please refer to Haque et al. (2020).