Bacillus cereus sensu lato is a facultative anaerobic gram-positive bacterium that can persist and survive in various conditions, including extreme ones. Due to its widespread distribution, these bacteria are not only a cause of food poisoning, but also a potential source of severe clinical infections. Hemolysin II (HlyII), belonging to the group of β-pore-forming toxins, is one of the key pathogenic factors of B. cereus sensu lato. The aim of the study was to develop a test system for detecting B. cereus hemolysin II. Materials and methods. A method for detecting HlyII was developed using a sandwich enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies against separate regions of HlyII. Validation parameters of the test system were determined according to international standards. Statistical analysis was performed using Excel 2016 and Statistica 7 software; regression analysis, using CurveExpert Professional. Results. The test-system for detecting hemolysin II with a detection range of 6.25 to 200 ng/mL was developed. The developed method enables the detection of the toxin from various B. cereus strains in the blood serum and culture fluid. Using the example of cultivating the natural B. cereus strain ATCC 14579, the suitability of the developed test system for assessing HlyII content in the culture fluid was confirmed. At the same time, the toxin was detected in the logarithmic growth phase of bacteria at temperatures of 25 and 30°C. Conclusions. Increased expression of the hlyII gene was described for clinical infections, making timely detection of this toxin important. A test system for detecting hemolysin II, which significantly affects a potential pathogenicity and clinical picture of the disease, will facilitate the diagnosis of the infection, as well as improve the infection control.