Studies show that hepatitis B virus (HBV) DNA isolation methods vary in their efficiency to extract DNA from serum samples. The purpose of the present study was to develop an improved method for isolation of HBV DNA and compare it with commonly used HBV DNA isolation protocols. In order to develop HBV DNA isolation protocol, serum samples were collected from patients and screened for the presence of hepatitis B surface antigen, hepatitis B e antigen and HBV DNA. Highly viremic samples were pooled and used to compare commonly used HBV DNA isolation methods; namely alkaline lysis, microwave treatment, organic, inorganic with modified inorganic method. DNA isolated by these methods was detected qualitatively by polymerase chain reaction and quantitatively with competitive polymerase chain reaction (cPCR). The modified inorganic method gave maximum yield of HBV DNA followed by inorganic, organic, microwave treatment and alkaline lysis method. Our data also demonstrated a critical role of proteinase K in HBV DNA isolation. DNA isolation method described here, in combination with a reproducible and sensitive quantitative technique would further help in accurate classification of HBV infected patients, designing suitable drug regimen for treatment and monitoring antiviral treatment as well as emergence of drug resistant mutants.