Reverse-phase high-performance Liquid chromatography (RP-HPLC) is used to characterize triple helical structures of synthetic collagen analogs. This technique is based on the different retention times exhibited by the triple helical conformations and the non triple helical structures formed by the same collagen analog. The difference in retention times is mainly caused by the change of hydrophobicity after triple helix formation. For synthetic collagen analogs composed of tripeptide sequences which do not possess hydrophilic groups in their side chains, such as the Gly-Pro-Pro and Gly-Pro-Nleu (Nleu represents N-isobutylglycine residue) sequences, the triple helical conformations possess longer retention times than the non triple helical structures. On the other hand, for collagen analogs composed of Gly-Pro-Hyp sequences which have hydrophilic groups in the side chains, the triple helical conformations elute at shorter retention times than the non triple helical structures. In reverse-phase HPLC profiles, the template-assembled analog, KTA-[Gly-(Gly-Pro-Nleu)(9)-NH2](3) (KTA represents cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid), exhibits one single peak which arises from the triple helical conformation. The HPLC profile of Ac-(Gly-Pro-Nleu)(9)-NH2 is compared to that of KTA- [Gly-(Gly-Pro-Nleu)(9)-NH2](3) to establish its triple helical HPLC peak. Both peaks from triple helical and non triple helical structures are observed in the HPLC profiles of single-chain collagen analogs Ac-(Gly-Pro-Nleu)(9)-NH2, (Gly-Pro-Pro)(10), and Ac-(Gly-Pro-Hyp)(9)-NH2. Under appropriate conditions, we demonstrate that the peaks associated with the triple helical structures are interconvertable with the peaks from the non triple helical structures.