We describe the discovery, development and in vivo activity of small molecules that inhibits HCV replication via direct interaction with the viral NS4B protein. The inhibitors were identified through a phenotypic, cell based, high throughput screen using the HCV subgenomic replicon. Compounds were then optimized to extremely high potency and pharmacokinetics. Mechanistic data generated suggests a hypothesis wherein the compounds described function by binding to NS4B, preventing the formation of the characteristic HCV induced sub‐cellular membranous web required for viral replication. Finally, in vivo proof of mechanism was established by employing a chimeric “humanized” mouse model of HCV infection to demonstrate for the first time that a small molecule with high in vitro affinity for NS4B can inhibit viral replication in vivo.