Hepatitis B virus (HBV) infection induces changes in the host cell proteome. Using label-free differential proteomics, we investigated changes in protein levels in HepG2NTCP hepatoma cells and primary human hepatocytes (PHH) following in vitro HBV infection. Our analysis revealed significant differences between these hepatocyte cell culture models, highlighting the importance of HBV inoculum composition. The actual response to active HBV replication, as observed in the proteome and secretome of PHH, involves specific changes in cellular pathways.