Hepatitis B virus resistance to antivirals is conferred by the selection of polymerase gene mutants with reduced susceptibility to nucleoside analogues. Because of the increasing number of resistant mutants to the different available drugs, several phenotypic assays have been developed to provide cross-resistance data to guide clinicians in the management of therapy. Their possible use in the routine follow-up of antiviral treatment is also discussed. The different in vitro models for the study of HBV resistance include enzymatic and cell-based assays. Their relevance for the analysis of viral fitness, drug susceptibility, and cross-resistance is also reviewed.