Inhibitors of the hepatitis C virus (HCV) NS5A protein are a key component of effective treatment regimens, but the genetic heterogeneity of HCV has limited the efficacy of these agents and mutations lead to resistance. We directly compared the efficacy of all clinically relevant NS5A inhibitors against HCV genotype 1–7 prototype isolates and resistant escape variants, and investigated the effects of pre-existing resistance-associated substitutions (RAS) on HCV escape from treatment.