The objective of this research study was to optimize fermentation parameters and the preservative agent for the production of acid milk with Lactobacillus acidophilus. Milk inoculated with L. acidophilus was incubated at 42, 45, and 50 °C for 8, 10, and 12 hours. The best fermentation parameters were observed with 108 UFC/mL at 45°C for 8 hours, obtaining a final pH of 4.5 and a titratable acidity of 90°D. The best preservative agent for sour milk stored without refrigeration was sodium benzoate (0.1%) with 98% acceptance. The best preservative agents for refrigeration were sodium benzoate (0.1%) and potassium sorbate (0.1%) with 100% acceptance in peach and custard apple flavors. Both preservatives showed a shelf life of 35 days and a microbial load of 109 UFC/mL. It is concluded that the optimization method developed here succeeded in elaborating acid milk inoculated with L. acidophilus that meets the standards for human consumption and as a probiotic drink.