Hydroxyproline is a non-essential amino acid found in collagen and few other extracellular animal proteins. It’s two isomeric forms trans-4-hydroxy-L-proline and trans-3-hydroxy-L-proline play a crucial role in collagen synthesis and thermodynamic stability of the triple-helical conformation of collagen and associated tissues. Various abnormalities in hydroxyproline metabolism have been shown to play key roles in the pathophysiology and pathogenesis of different diseases. The elevated level of hydroxyproline is observed in several disorders, e.g., graft versus host disease, keloids, and vitiligo while its decreased level is a marker of poor wound-healing. This review explores the potential of using hydroxyproline as a biochemical marker to understand the pathogenesis, molecular pathophysiology and treatment of these diseases. The review concludes with an outlook on the scope and challenges in the clinical implementation of hydroxyproline as a biomarker.