Amphibian skin secretions are rich sources of biomolecules with diverse biotechnological applications. In this study, the general skin secretions of Bombina variegata is suggested as an alternative source of collagenolytic enzymes applied in different areas of industry such as the pharmaceutical, textile and leather sectors. Collagenolytic proteases were partially purified from skin secretions by ion-exchange chromatography on a DEAE-Sepharose column and several methodological approaches were tested to optimize the purification protocol. The salt concentration, and pH of mobile phase were varied to determine their effects on the resolution of the collagenolytic enzyme separation. Thus, time and resolution of purification were optimized yielding the required purity and amounts of active enzymes that can be useful for several research areas and biotechnology.