Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity that can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties were used as decisive criteria for further application of the enzymes, with emphasis on minimal destruction of protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAGs content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective, alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogenous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to that on pure rat tail collagen type I gels. Papain is not just highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.