This invention provides efficient HCV replicase complexes comprising novel RNA template and primer pair. Assay systems are also provided, which use such complexes, for detecting replicase activity, quantitatively studying the kinetics and mechanism of HCV NS5B-catalyzed nucleotide incorporation, and identifying inhibitors of HCV replicase. The assay systems use small and well-defined synthetic RNAs which allow efficient assembly of all catalytic components in the quaternary complex for HCV NS5B-directed RNA replication. Specific template-primer requirements for efficient RNA synthesis by HCV NS5B replicase are provided for use in assay systems.