The book "A closer look at proteolysis" is conceived as a review of modern knowledge from various fields related to proteolytic processes. Due to the simple approach to explaining various aspects of the topics related to proteolysis, it will be of interest to a wide audience, as well for the scientific community. The first chapter provides an overview of basic concepts related to proteolysis, and the classification of proteases with an emphasis on proteases that have been identified as key in various pathophysiological conditions. After that, in the second chapter, the reader is introduced to proteases that participate in the development of malignancy. The proteases that are perceived as potential candidates for the development of anticancer treatments are highlighted. The third chapter provides an insight into the basics of intracellular proteolysis, with an emphasis on proteolysis of the intrinsically disordered proteins and the consequences that proteolysis of these proteins has in plant cells particularly. In the fourth chapter, the authors provide the latest knowledge on the possibility of using a standard protease for mass spectrometry to determine post-translational modifications of proteins, which is the most recent challenge in proteomics-based research. Although often used as therapeutics, peptides and proteins are susceptible to proteolysis under physiological conditions, so the design of drugs that mimic the action of proteins and peptides is a big challenge for medicinal chemists. In the fifth chapter, the authors elaborate on the ways to overcome proteolysis in potential therapeutic agents, using the rational design of modern peptidomimetics. The authors of the sixth chapter look at the correlation between the stability to proteolytic digestion and the allergenicity of food proteins. Chapters seven and eight are devoted to microbial proteases. The author of the seventh chapter gives an overview of the yeast proteases characterized so far, while the authors of the eighth chapter consider the possibilities for the production of purification of Bacillus proteases and their application in various biotechnological processes. In the last, ninth chapter, the technological significance of proteolytic processes in the food industry is emphasized. The authors presented methodologies that can be used to determine different bioactive peptides produced during cheese ripening.