Cyclosporine A (CsA) is an immunosuppressive molecule that also impedes replication of hepatitis C virus (HCV). CsA inhibits isomerase activity of cellular-encoded cyclophilin proteins, of which cyclophilin A (CypA) in particular is required for HCV replication. Evidence suggests that the HCV-encoded NS5A and NS5B proteins may govern dependence of the virus on CypA-mediated isomerase activity, although the molecular mechanisms involved are unclear. However, association of NS5A and NS5B, with CypA has been reported, raising the possibility that direct interaction between these proteins facilitates HCV replication. In the present study, mammalian two-hybrid and AlphaLISA technologies were utilized to detect interactions between NS5A and NS5B, with CypA. AlphaLISA analysis revealed associations between NS5A and CypA using purified proteins, and in cell lysates prepared from co-transfected cells. Importantly, the NS5A-CypA interactions were sensitive to CsA in a dose-responsive manner and an isomerase mutant of CypA interacted with NS5A less efficiently than wild-type CypA. These findings correlate the anti-HCV properties of CsA with an ability of the compound to disrupt NS5A-CypA interactions in vitro and in vivo, whilst providing the basis for development of assay platforms suitable to screen compound libraries for novel inhibitors of the NS5A-CypA interaction.