Hepatitis C virus (HCV) infection affects more than 170 million people world-wide and can progress to chronic disease resulting in liver cirrhosis and hepatocellular carcinoma. The high genetic variability of HCV and the rapid development of drug-resistant strains during treatment have fuelled a search for new direct-acting antiviral agents. One recently approved class of agents are believed to target the HCV protein NS5A, which plays a critical but poorly defined role in reproduction of the viral RNA genome, although precisely where these compounds interact and how they affect NS5A function is unknown. We have developed an in vitro assay based on microscale thermophoresis allowing us to demonstrate that two clinically relevant NS5A inhibitors bind tightly (low nanomolar affinity) to NS5A domain 1 and inhibit RNA binding (1). Conversely, prior RNA binding inhibits compound binding. By combining these results with crystallographic and molecular modelling studies, we propose a mechanism for NS5A inhibitors in which they act by favouring a dimeric structure of NS5A that does not bind RNA.