Proteoglycans are high molecular weight glycoproteins with potential benefits in preventing osteoarthritis, reducing inflammation, enhancing immune function, and promoting skin health. Aggrecan, a key proteoglycan with glycosaminoglycan (GAG) chains, poses challenges in accurate quantification due to its complex structure. We hypothesize that by selecting target peptides from core proteins that exclude post-translational modifications such as GAG attachment, proteoglycans can be analyzed with high sensitivity and accuracy. In this study, we identified aggrecan from salmon nasal cartilage (Oncorhynchus keta) using liquid chromatography-mass spectrometry (LC-MS). We developed a quantitative multiple reaction monitoring (MRM)-MS assay with stable-isotope-labeled peptides. This method demonstrated high precision and sensitivity, achieving a limit of detection (LOD) of 0.0008 μg/mL and a lower limit of quantification (LLOQ) of 0.0025 μg/mL. These findings validate that targeting core proteins enables accurate proteoglycan quantification and support its application in quality assessment and development of salmon nasal cartilage-based foods.