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Nagel O.G., Molina M.P., Althaus R.L. (2013): Use of chemometric techniques to design a microbiological method for sulfonamide detection in milk. Czech J. Food Sci., 31: 627–632. We proposed an experimental design of a microbial bioassay of dichotomous response (positive or negative) using Bacillus subtilis BGA for the detection of sulfonamide residues. In the first stage, the bioassay response time was reduced to 6 h by increasing the spore concentration of B. subtilis . Then, the effects of spore, indicator, trimethoprim (TMP) concentration, and volume of the culture medium were examined with a Plackett Burman design (2 4-1 ). Finally, the effect of TMP concentration on the method detection capabilities and specificity was analysed using a logistic model with interaction. The detection capabilities of sulfonamides in milk are close to the MRLs when using 500 mg/l of TMP in the culture medium of the bioassay. It is concluded that the experimental design techniques and a logistic regression model can be used to design successfully a dichotomous response bioassay.