Little is known regarding radiation-induced matrikines and the possible degradation of extracellular matrix following therapeutic irradiation. The goal of this study was to determine if irradiation can cut collagen proteins at specific sites, inducing potentially biologically active peptides against cartilage cells. Chondrocytes cultured as 3D models were evaluated for extracellular matrix production. Bystander molecules were analyzed in vitro in the conditioned medium of X-irradiated chondrocytes. Preferential breakage sites were analyzed in collagen polypeptide by mass spectrometry and resulting peptides were tested against chondrocytes. 3D models of chondrocytes displayed a light extracellular matrix able to maintain the structure. Irradiated and bystander chondrocytes showed a surprising radiation sensitivity at low doses, characteristic of the presence of bystander factors, particularly following 0.1 Gy. The glycine-proline peptidic bond was observed as a preferential cleavage site and a possible weakness of the collagen polypeptide after irradiation. From the 46 collagen peptides analyzed against chondrocytes culture, 20 peptides induced a reduction of viability and 5 peptides induced an increase of viability at the highest concentration between 0.1 and 1 µg/ml. We conclude that irradiation promoted a site-specific degradation of collagen. The potentially resulting peptides induce negative or positive regulations of chondrocyte growth. Taken together, these results suggest that ionizing radiation causes a degradation of cartilage proteins, leading to a functional unbalance of cartilage homeostasis after exposure, contributing to cartilage dysfunction.