Chronic HBV infection leads to an estimated 820,000 deaths yearly. It has no cure and new treatments need to be developed. APOBEC3 proteins can restrict the replication of viruses (e.g., HIV) and retrotransposons. This study aimed to elucidate the role of APOBEC3 proteins in Hepatitis B virus replication. It was observed that APOBEC3G proteins colocalise with HBV polymerase proteins in areas of the cytoplasm using immunofluorescence. It was shown that USF1 could upregulate APOBEC3 gene expression and that USF1 enhanced HBV replication. qPCR used to measure levels of APOBEC3 mutated viruses that do not survive the mutations in HepG2.2.15 cells, these results showed that APOBEC3G and APOBEC3DE could inhibit extracellular virion-associated HBV DNA levels by 48% and 16% respectively and could inhibit cytoplasmic HBV DNA by 65% and 39% respectively. The numbers and percentages of G to A mutations in the HBV X region detected by Phusion U polymerase-based 3D-PCR (modified 3D-PCR) were lower than those detected by Taq polymerase-based 3D-PCR (3D-PCR) in empty vector and APOBEC3G vector transfected HepG2.2.15 cells. However, numbers and percentages of G to A mutations in the HBV X region detected by modified 3D-PCR were higher than those detected by 3D-PCR in APOBEC3DE transfected HepG2.2.15 cells. Primer-ID NGS results demonstrated a higher number of G to A and C to U mutations in the HBV S region compared to the HBV X region. Primer-ID NGS also showed that these mutations already existed in the HBV pre-core, X, and S regions in HepG2.2.15 cells that were not transfected with APOBEC3 plasmids. These novel approaches may help to measure more accurately the effects of APOBEC3 proteins on the HBV virus in vitro and in vivo and could be important to evaluate the possible role of APOBEC3 proteins in treatments for HBV.