Molecular biology techniques are routinely used to diagnose and monitor treatment of patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. These tools can detect and quantify viral genomes and analyze their sequence to determine their genotype or subtype and to identify nucleotide or amino acid substitutions associated with resistance to antiviral drugs. They include real-time target amplification methods, which have been standardized and are widely used in clinical practice to diagnose and monitor HBV and HCV infections, and next-generation sequencing techniques, which are still restricted to research laboratories. In addition, new enzyme immunoassays can quantify hepatitis B surface and hepatitis C core antigens, and point-of-care tests and alternatives to biologic tests that require whole-blood samples obtained by venipuncture have been developed. We review these new virologic methods and their clinical and research applications to HBV and HCV infections.