[EN] Aims: 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). Methods and Results: In a first stage, the response time of bioassay was reduced to 5h when the logarithm of spore concentration (log S) was increased. Later, a Plackett Burman design (2 6-3) 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¿gl -1) and oxytetracycline (100¿gl -1) were adjusted to their MRLs through the desirability function. Conclusions: 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¿gl -1. Significance and Impact of the Study: 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. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology. This research has been carried out as part of the AGL2003-03663 project and has been financially assisted by the Spanish Ministry of Education and Science (Madrid, Spain). The authors thank the Polytechnic University of Valencia (UPV, Spain) for funding Dr. Rafael Althaus's collaboration with the Institute for Animal Science and Technology (UPV). Zorraquino Lozano, MÁ.; Althaus, RL.; Roca Marugán, MI.; Molina Pons, MP. (2011). Heat Treatment Effects on the Antimicrobial Activity of Macrolide and Lincosamide Antibiotics in Milk. Journal of Food Protection. 74(2):311-315. doi:10.4315/0362-028X.JFP-10-297