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More than 300 million people worldwide are diagnosed with a chronic hepatitis B virus (HBV) infection. Nucleos(t)ide viral polymerase inhibitors are available on the market and can efficiently treat patients with chronic HBV. However, life-long treatment is needed as covalently closed circular DNA (cccDNA) persists in the hepatocyte nucleus. Hence, there is a high demand for novel therapeutics that can eliminate cccDNA from the hepatocyte nucleus and cure chronically infected HBV patients. The gold standard for in vitro HBV studies is primary human hepatocytes (PHHs). However, alternatives are needed due to donor organ shortage and high batch-to-batch variability. Therefore, human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are being explored as an in vitro HBV infection model. We recently generated hPSC lines that overexpress three transcription factors (HC3x) and that, upon differentiation in a high amino-acid supplemented maturation medium, generate a more mature hepatocyte progeny (HC3x-AA-HLCs). Here, we demonstrate that HBV can efficiently infect these HC3x-AA-HLCs, as was shown by the presence of HBV core (HBc) and surface antigens. A clear increasing release of HBV surface and e antigens was detected, indicating the formation of functional cccDNA. Moreover, back-titration of culture supernatant of HBV-infected HC3x-AA-HLCs on HepG2-NTCP cells revealed the production of novel infectious HBV particles. Additionally, an increasing number of HBc-positive HC3x-AA-HLCs over time suggests viral spreading is occurring. Finally, the HC3x-AA-HLC model was validated for use in antiviral drug studies using the nucleoside reverse-transcriptase inhibitor, lamivudine, and the HBV entry inhibitor, Myrcludex B.