Collagen provides mechanical and biological support for virtually all human tissues in the extracellular matrix (ECM). Its defining molecular structure, the triple-helix, could be damaged and denatured in disease and injuries. To probe collagen damage, the concept of collagen hybridization has been proposed, revised, and validated through a series of investigations reported as early as 1973: a collagen-mimicking peptide strand may form a hybrid triple-helix with the denatured chains of natural collagen but not the intact triple-helical collagen proteins, enabling assessment of proteolytic degradation or mechanical disruption to collagen within a tissue-of-interest. Here we describe the concept and development of collagen hybridization, summarize the decades of chemical investigations on rules underlying the collagen triple-helix folding, and discuss the growing biomedical evidence on collagen denaturation as a previously overlooked ECM signature for an array of conditions involving pathological tissue remodeling and mechanical injuries. Finally, we propose a series of emerging questions regarding the chemical and biological nature of collagen denaturation and highlight the diagnostic and therapeutic opportunities from its targeting.