ObjectiveTo compare the efficacy of Dixon and Mood up-and-down method and Karber method in determining tetanus toxin LD₅₀ in mice. Methods Tetanus toxin in a dose of 0.5 mL at concentration of 0.9,0.1,0.5,0.0,0.267 or 0.256 ng/mL was subcutaneously injected into NIH mice. The LD₅₀ of tetanus toxin was determined by Dixon and Mood up-and-down procedure and Karber test. ResultsThe LD₅₀ of tetanus toxin was 4.97 ng/kg body weight measured by Karber method, and 4.93 ng/Kg body weight by the up-and-down procedure. Conclusions Both Dixon and Mood up-and-down procedure and Karber test can achieve the same LD₅₀ of tetanus toxin, but the up-and-down procedure is more simple and convenient, and 8 animals are enough for this test.