The aim of this study was to compare the clinical applicability of quantitative serum hepatitis B surface antigen (HBsAg), hepatitis B e-antigen (HBeAg) and hepatitis B virus (HBV) DNA for predicting virological response (VR) to pegylated interferon (PEG-IFN) therapy. Thirty HBeAg-positive chronic hepatitis B patients who received PEG-IFN-alpha-2b for 48 weeks were enrolled. Quantitative HBsAg, HBeAg and HBV DNA were measured before, during and after the therapy. Paired liver biopsies were performed before and after treatment for covalently closed circular (ccc)DNA and intrahepatic HBV DNA analysis. VR at 48 weeks post-treatment, defined as HBeAg seroconversion and HBV DNA less than 10 000 copies/mL was achieved in 10 (33.3%) patients. Responders had significantly lower baseline HBsAg, HBeAg, cccDNA and intrahepatic HBV DNA levels than non-responders. Baseline and reduced levels of log(10) HBsAg and log(10) HBeAg correlated well with those of log(10) cccDNA and log(10) total intrahepatic HBV DNA. Responders showed consistent decrease in serum HBsAg, HBeAg and HBV DNA levels during therapy. HBeAg level of 2.0 log(10) sample to cut-off ratio at week 24 on therapy provided the best prediction of sustained virological response, with sensitivity and negative predictive values of 85% and 92%, respectively. One patient (3.3%) who cleared HBsAg at follow up exhibited a more rapid decline in serum HBsAg during therapy than those who developed VR without HBsAg clearance. Quantitative measurement of serum HBeAg during therapy may be superior to serum HBsAg and HBV DNA as a prediction of HBeAg seroconversion. Kinetics of HBsAg levels on therapy may help predict HBsAg clearance after treatment.