Dibenzocyclooctynes have emerged as promising scaffolds for bioorthogonal ligation. An important structural aspect that has not been addressed so far relates to their chirality. Herein, we explore, by theoretical and experimental methods, this structural aspect that has been neglected so far. First, computational analysis is conducted, and the results are used as a guide for the experimental investigation. Next, an array of different experiments (high-performance liquid chromatography (HPLC) on chiral columns, chiroptical spectroscopy, and X-ray diffraction) for structure elucidation is scrutinized in concert. Finally, this work demonstrates the chirality and the stereodynamic behavior of dibenzocyclooctynes and their triazole derivatives with simple azides and also uncovers their conformational behavior.