One of the main biochemical changes that take place during the processing of dry-cured ham is the degradation of the muscle protein fraction, mainly due to the action of muscle enzymes. In the present study, the isolation and tentative identification of 137 fragments from myosin light chain 1 (MLC 1), together with 88 fragments originated from myosin light chain 2 (MLC 2), have been achieved for the first time in Spanish dry-cured ham, proving the intense proteolysis experienced by myofibrillar proteins after dry-cured processing. This study was carried out by use of proteomic technology for peptide identification, and the possible enzymes contributing to the degradation of these proteins were also further discussed.