When slaughtering and processing poultry, large quantities of meat by-products are generated; therefore, the development of the newest methods for processing secondary raw materials is an urgent problem. Animal proteins have relevant technological applications and are also considered as a potential source of bioactive peptides. Current technologies suggested that protein substances can be isolated from meat co-products through microbial hydrolysis. The purpose of the study was to optimize the technological parameters of microbial hydrolysis of hen combs and to analyze the modification of the microstructure and properties of hydrolyzed by-products under the action of bacterial enzymes. Hen’s combs were hydrolyzed by bifidobacteria and concentrated Propionix liquid. A multifactorial experiment was used to determine the optimal conditions for the hydrolysis process. As a result of the study, multiple regression equations and response surfaces were obtained, which describe the process of hydrolysis of hen combs to identify the optimal hydrolysis parameters. Temperature, amount of bacterial concentrate and hydrolysis period are factors that have a significant impact on the degree of hydrolysis. The results of microscopic and dispersed analysis confirm the good hydrolyzability of combs due to changes in structural components and an increase in the amount of smaller protein particles.