HBV is a small, enveloped DNA virus that replicates by reverse transcription via an RNA intermediate. Current anti-HBV treatment regiments that include interferon α and nucleos(t)ide analogs have insufficient efficiency, are of long duration and can be accompanied by systemic side effects. Though HBV RNaseH is essential for viral replication, it is unexploited as a drug target against HBV. RNaseH inhibitors that actively block viral replication would represent an important addition to the potential new drugs for treating HBV infection. Here we describe two methods to measure the activity of RNaseH inhibitors. DNA oligonucleotide-directed RNA cleavage assay allows low-throughput screening of compounds for potential anti-HBV RNaseH activity in vitro. Analysis of preferential inhibition of plus-polarity DNA strand synthesis by HBV RNaseH inhibitors in a cell culture model of HBV replication can be used to validate the efficiency of these compounds to block viral replication.