The proteolytic system plays a crucial role in maintaining the homeostasis and defence against pathogens. Its proper functioning depends on the balance between the activities of proteases and their inhibitors. The disturbing of this balance, caused, for example, by Varroa destructor, brings about physiological/metabolic changes leading to premature aging. Therefore, our study aimed to investigate the effect of V. destructor on the activities of acidic, neutral, and alkaline proteases and their inhibitors in bee hemolymph and fat body segments (from tergite 3, tergite 5 and sternite). The parasite caused a decrease in the protease and protease inhibitor activities, accelerating the aging process. In healthy worker bees, proteolytic activity in the fat body segments increased with age, peaking at 21–28 days, and subsequently declined in 35-day-old workers. Additionally, it was observed that tergite 5 was the segment characterized by the highest activity of the proteolytic system, which indicates that it can be used as a biomarker of aging and immunity. Studying the proteolytic system is important because it allows for a more detailed understanding of immunity mechanisms, aging processes, and responses to infection, which may contribute to the development of preparations promoting apian health