Bioactive peptides derived from food matrices are currently a subject of intensive research， due to the multiple health benefits they can exert in the human body. However， the small size and low abundance of these peptides in complex matrices as well as the effect of food processing and gastrointestinal digestion on their structure， sequence， and functionality， are important challenges. Spanish dry-cured ham has been reported as a good and natural source of bioactive peptides mainly showing antihypertensive and antioxidant activities. In this work， the antioxidant peptide AEEEYPDL， identified from 9 months of curing dry-cured ham， showed good resistance to different heat treatments and salt contents. The bioactivity of the peptide significantly decreased after simulated gastrointestinal digestion. An optimised multiple reaction monitoring methodology was able to quantify the peptide AEEEYPDL at a concentration of 0.148 fg per g of dry-cured ham， despite the existing challenges in the quantification of bioactive peptides from complex matrices.