During a screening of cyclodextrins (CDs) as chiral selectors for the separation of daclatasvir (DCV) and its enantiomer by capillary electrophoresis (CE), an unusual phenomenon for CDs was observed, that is two peaks with a plateau in between using γ-CD as chiral selector. The same result was encountered when enantiopure DCV was injected or when analyzing a sample containing enantiopure DCV and γ-CD in a CD-free background electrolyte. Peak coalescence was observed at 45°C and at a pH above 3.5. Two peaks with a plateau were also observed for DCV stereoisomers as well as a structural analog. However, only a single peak was detected if one or both amino acid moieties of DCV were lacking. Nuclear magnetic resonance (NMR) experiments including Nuclear Overhauser effect-based methods showed that in solution DCV adopted a folded conformation in which the isopropyl side chain of the valine residues pointed toward the aromatic rings of DCV. Moreover, NMR unequivocally demonstrated the simultaneous formation of DCV-γ-CD inclusion complexes with 1:1 and 2:1 stoichiometry, which was corroborated by mass spectrometry. In both complexes, DCV also adopted a folded structure. The RSSR-diastereomer of DCV as well as an analog lacking one of the amino acid moieties also formed 1:1 and 2:1 complexes with γ-CD although a plateau was only observed in the case of the RSSR-diastereomer. As shown by CE-MS, both DCV-γ-CD complexes surprisingly comigrated as the first peak, while the second migrating peak represents non-complexed DCV. Copyright © 2020. Published by Elsevier B.V.