Consumption of various dry-cured meat products, especially dry-cured ham is among most popular eating habits in many European countries, especially in the Mediterranean region. Istrian dry-cured ham is a high quality product of exceptional economic importance for Croatia which is produced according to a set of guidelines strictly defined in the Specification designation of origin. Premises for the product constant and defined quality include knowledge production of the Istrian dry-cured ham, process optimisation as well as quality assurance. These ca be used in prevention of possible customer frauds. In this light, it is necessary to develop new analytical protocols and platforms for quality control such as for example those based on proteomics methods and approaches. During the dry-cured ham production process, it was found that muscle proteins undergo extensive proteolysis and key enzymes involved in these processes have been studied in details with the aim to understand the mechanisms of variability in the quality of final product. As a result of proteolytic cleavage during the rippening process, a specific set of proteins and peptides occur whose composition is directly dependent on the quality of raw meat, production process (drying and/or smoking, ripening) and recipes. Therefore, a new proteomics protocol for analysis of Istrian dry-cured ham proteolytic profile is developed within presented research by use of gel-electrophoresis coupled with matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). The results showed a characteristic proteolityc profile of the Istrian dry-cured ham that include actin, myosin, beta chain of tropomyosin and alpha-1 chain of tropomyosine proteins and protein fragments.