Osteoarthritis (OA) is a pathology characterized by the loss of articular cartilage. In this study, we performed a peptidomic strategy to identify endogenous peptides (neopeptides) that are released from human osteoarthritic tissue, which may serve as disease markers. With this aim, secretomes of osteoarthritic and healthy articular cartilages obtained from knee and hip were analyzed by shotgun peptidomics. This discovery step led to the identification of 1175 different peptides, corresponding to 101 proteins, as products of the physiological or pathological turnover of cartilage extracellular matrix. Then, a targeted multiple reaction monitoring-mass spectrometry method was developed to quantify the panel of best marker candidates on a larger set of samples (n=62). Statistical analyses were performed to evaluate the significance of the observed differences and the ability of the neopeptides to classify the tissue. Eight of them were differentially abundant in the media from wounded zones of OA cartilage compared to the healthy tissue (p<0.05). Three neopeptides belonging to Clusterin and one from Cartilage Oligomeric Matrix Protein showed a disease-dependent decrease specifically in hip OA, whereas two from Prolargin (PRELP) and one from Cartilage Intermediate Layer Protein 1 were significantly increased in samples from knee OA. The release of one peptide from PRELP showed the best metrics for tissue classification (AUC=0.834). The present study reveals specific neopeptides that are differentially released from knee or hip human osteoarthritic cartilage compared to healthy tissue. This evidences the intervention of characteristic pathogenic pathways in OA and provides a novel panel of peptidic candidates for biomarker development.