We here developed new techniques that substantially improve cccDNA detection and quantification, including a sample pretreatment method that i) exploits high temperature and exonuclease V (an ATP-dependent, bidirectional exonuclease) digestion to effectively increase the amplification efficiency of HBV cccDNA by removing rcDNA and denaturing the cccDNA template, and ii) a method that splits cell samples and uses separate extraction technologies to facilitate "host normalization" based on host genomic DNA signals.