C-telopeptide of type II collagen (CTX-II) has been shown to be a highly relevant biomarker of cartilage degradation in human rheumatic diseases， if measured in synovial fluid or urine. However， serum or plasma CTX-II have not been demonstrated to have any clinical utility to date. Here， we describe the GPDPLQ ELISA which targets the EKGPDPLQ↓ neo-epitope， an elongated version of the CTX-II neo-epitope (EKGPDP↓)， speculated to be a blood-precursor of CTX-II generated by the cysteine protease cathepsin K. Human osteoclast cartilage resorption cultures as well as oncostatin M and tumour necrosis factor α-stimulated bovine cartilage explant cultures were used to validate GPDPLQ biologically by treating the cultures with the cysteine protease inhibitor E-64 and/or the matrix metalloproteinase (MMP) inhibitor GM6001 to assess the potential contributions of these two protease classes to GPDPLQ release. Cartilage resorption-derived GPDPLQ release was inhibited by E-64 (72.1% inhibition)， GM6001 (75.5%)， and E-64/GM6001 (91.5%)， whereas CTX-II release was inhibited by GM6001 (87.0%) but not by E-64 (5.5%). Cartilage explant GPDPLQ and CTX-II release were both fully inhibited by GM6001 but were not inhibited by E-64. No clinically relevant GPDPLQ reactivity was identified in human serum， plasma， or urine from healthy donors or arthritis patients. In conclusion， the GPDPLQ biomarker is released during osteoclast-derived cysteine protease- and MMP-mediated cartilage degradation in vitro， whereas CTX-II release is mediated by MMPs and not by cysteine proteases， as well as from MMP-mediated cartilage degradation under a pro-inflammatory stimulus. These findings suggest that GPDPLQ may be relevant in diseases with pathological osteoclast activity and cartilage degradation. Further studies are required to validate the neo-epitope in human samples.