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In this study, a microbiological method of dichotomous response using Bacillus cereus was designed and optimized to detect tetracyclines (TCs) at concentrations near to the maximum residue limits (MRLs).In a first stage, the response time of bioassay was reduced to 5 h when the logarithm of spore concentration (log S) was increased. Later, a Plackett Burman design (26–3) was analysed using logistic regression model. This design indicates significant effects of log S and chloramphenicol (CAP) on the detection limit (DL) of TC. Then, the response surfaces (RS) of the TCs DTs as a function of log S and CAP were plotted using a Dohlert design and the logistic regression model. These RS show a linear decrease with the raise of CAP and a quadratic effect of log S. Finally, the DTs of TC (109 μg l−1) and oxytetracycline (100 μg l−1) were adjusted to their MRLs through the desirability function.By successive application of experimental design techniques could be optimized a bioassay for the detection of TC residues in milk. The best conditions have been achieved when the assay was made with log S = 5·12 and CAP = 470 μg l−1.Experimental design techniques together with the logistic regression model and the desirability function represent an adequate tool for the optimization of a bioassay with binary response.