Proteolysis is a process where proteins are broken down into smaller polypeptides or amino acids, comprising one of the important posttranslational modifications of proteins. Since this process is exquisitely achieved by specialized enzymes called proteases under physiological conditions, abnormal protease activity and dysregulation of their substrate proteins are closely associated with a progression of several neurodegenerative diseases including Alzheimer disease, Parkinson disease, stroke, and spinal cord injury. Thus, it is important to identify the specific substrates of proteases with nonbiased high-throughput screenings to understand how proteolysis contributes to neurodegeneration. Here, we described a so-called gel-based protease proteomic approach. Critical steps of our novel strategy consist of two-dimensional polyacrylamide gel electrophoresis (2-DE)-based protein separation and in vitro incubation with the specific protease of interest. As a prototypic example, cellular lysates obtained from neuronal cells are separated by an isoelectric focusing, and the resulting immobilized proteins on a gel strip are incubated with a predetermined amount of a recombinant or a purified protease. By densitometric analysis of the Coomassie Brilliant Blue-stained gel images following separation by 2-DE, significantly altered protein spots are subjected to a mass spectral analysis for protein identification. Interestingly, the concepts of our strategy can be applied to any proteases, and to any neural cells or neural tissues of one’s interest. Since the immobilized protein spots are exposed to the purified protease, this protocol ensures the identification of only substrates that are directly cleaved by specific protease. This protocol ensures to avoid the possibility of identifying substrates that may be cleaved by combinatorial or sequential activation of proteolytic enzymes present in a liquid state of the lysates. We propose that our strategy can be effectively utilized to provide meaningful insights into newly identified protease substrates and to decipher molecular mechanisms critically involved in neurodegenerative processes.