Cyclosporine A (CsA) and its chemical analogues EthVal4Cs, MeVal4Cs, and Me(D-Ala)3EthVal4Cs (Alisporivir) all interact with cyclophilin A (CypA). The latter Alisporivir is a nonimmunosuppressive CsA derivative that has potent anti-HCV properties in clinical trials. We show here that NMR spectroscopy can be used to rank this series of related pharmacological molecules despite their high affinity for the target protein and low solubility in water. The novel method is based on the possibility to detect distinct NMR signals from the different protein complexes in a mixture. The method has enabled us to distinguish subtle effects of discrete chemical modifications of the parent molecule on the affinity of the ligands for the target protein.