Osteoarthritis (OA) is caused by the degradation of articular cartilage and affects approximately 80% of people over the age of 65. Matrix metalloproteinases (MMPs) belong to a group of zinc endopeptidases that degrade extracellular matrix (ECM) proteins in cartilage. MMP-13, also known as collagenase 3, cleaves type II collagen more rapidly than other MMPs and therefore is an important target for the treatment of OA. The lipid peroxidation product 4-hydroxy-2-(E)-nonenal (HNE), generated under oxidative stress, is known to play a crucial role in cartilage degradation; however, the mechanism is not yet fully understood. An approach has been developed to monitor HNE modification sites by incubating rhMMP-13 ± HNE in vitro followed by analysis of tryptic digests by UHPLC coupled to high resolution (HR) quadrupole-time-of-flight (QqTOF) tandem mass spectrometry (MS/MS). The analysis elucidated several covalently modified histidine and cysteine residues. The reaction was monitored using different HNE concentrations and incubation times. A targeted assay, using multiple-reaction monitoring (MRM), was then optimized to increase the sensitivity of detecting these modification sites in biological samples. HNE-related covalent modifications of MMP-13 were confirmed in enriched extracts from interleukin 1β-activated chondrocytes from OA patients using HR-MS/MS and MRM analysis.