During milk fermentation with lactic acid bacteria (LAB) can be produced biologically active peptide sequences known as bioactive peptides. One of the most important biological activities is angiotensin-converting enzyme (ACE) inhibitory activity. The ACE- inhibitory activity depends on the fermentations conditions (temperature, pH, and inoculum). To evaluate the effect of some fermentation conditions on ACE-inhibitory activity a central composite design was used. Previously, 3 probiotic strains Lactobacillus plantarum, L. pentosus and L. acidipiscis were isolated from double cream cheese produced in the state of Chiapas (Mexico). These probiotic bacteria were shown to generate an ACE-inhibitory activity (more than 50%) in vitro tests. Lactobacillus plantarum was chosen for evaluating the effect of temperature (X₁), initial pH (X₂) and inoculum concentration (X₃) on the generation of ACE- inhibitory activity (Y). This study aims to optimize the ACE-inhibitory activity during milk fermentation by Lactobacillus plantarum using response surface methodology (RSM). For the optimization of fermentation process, a central composite design was used. ACE- inhibitory activity (response variable) was measured by the Cushman and Cheung method at initial and final fermentation time (16 h). The equation for the proposed model and model parameters where calculated with NCSS 11 Data Analysis Software. The mathematical model for the generation of ACE-inhibitory activity of fermented milk with Lactobacillus plantarum was the following: Y = 524.99 − 122.70X₁ + 344.85X₂ + 186.79X₃ + 1.66X₁² − 32.33X₂² − 0.86X₃² + 7.47X₁X₂+7.81X₁X₃ − 89.00X₂X₃ − 0.100X₁²X₂− 0.10X₁²X₃ + 6.03X₂²X₃. The results of regression analysis showed that initial pH was the most important factor positively affecting the ACE-inhibitory activity. Other factors significantly affecting the activity were inoculum and temperature (negative correlation). This mathematical model predicted the ACE-inhibitory activity in 86.99% of the cases.