The histamine sensitization test is a widely used method for measuring the residual toxicity of pertussis toxin in acellular pertussis vaccines. Although it has been used as a routine assay for decades, the current protocols are difficult to standardize because the test results vary considerably and are based on several factors, including mouse strain, age and sex. In this study, we observed that mice of strains CD1, ddY and C57/BL6 were sufficiently sensitive to pertussis toxin among six mice strains tested and that aged male mice were more sensitive to pertussis toxin than younger or female mice. Using this animal model, we showed pertussis toxin dose-dependent responses in the two histamine sensitization test protocols based on either lethal end-point determination or mouse rectal temperature measurement. Sensitivity to pertussis toxin was further enhanced by the addition of lipopolysaccharide in both methods. With these improvements, pertussis toxin activity can be estimated more accurately and reproducibly using a reduced number of animals.