Density functional theory (DFT) has been applied to the analysis of interligand vibrations in two chiral isomers of hydroperoxo complex of cobalt bleomycin (BLM-Co-III-OOH, BLM = bleomycin). The DFT-based normal coordinate analysis reveals that O-16/O-18 isotope-sensitive modes associated with the Co-OOH moiety uniquely reflect the chiral organization of ligands around the cobalt atom. This study provides an independent probe of cobalt chirality coordinated to BLM and shows that interligand modes associated with the Co-OOH moiety could be used as a structural marker of the chiral isomers.