During treatment of chronic hepatitis B virus (HBV) infections, loss or seroconversion (SC) of the HBV surface antigen (HBsAg) is considered a functional cure. HBsAg consists of the large (LHBs), middle (MHBs), and small surface protein (SHBs) and their relative proportions correlate strongly with disease stage. Our aim was to assess the association between HBsAg composition and functional cure during treatment METHODS: A total of 83 patients were retrospectively analyzed. Seventeen of 64 patients achieved HBsAg loss during nucleos(t)ide analogue (NA) treatment and 3/19 patients following treatment with pegylated interferon-alfa2a (PEG-IFN) for 48 weeks. Sixty-three patients without HBsAg loss were matched as controls. LHBs, MHBs and SHBs were quantified in sera collected before and during treatment.Before treatment, median MHBs levels were significantly lower in patients with subsequent HBsAg loss than in those without (p=0.005). During treatment, MHBs and LHBs proportions showed a fast decline in patients with HBsAg loss, but not in patients with hepatitis B e antigen (HBeAg) SC only or patients without serologic response. MHBs became undetectable by month 6 of NA treatment in all patients with HBsAg loss, which occurred on average 12.8±8.7 (0-52) months before loss of total HBsAg. ROC analyses revealed that MHBs proportion was the best early predictor of HBsAg loss before NA treatment (AUC=0.726, p=0.019). In patients showing HBsAg loss with PEG-IFN, MHBs and LHBs proportions showed similar kinetics.Quantification of HBsAg proteins shows promise as a novel tool to predict early treatment response. These assessments may help optimize individual antiviral therapies aiming to increase the rates of functional cure in chronically HBV-infected patients.