Calcium gluconate (0.0 to 0.5%), sodium gluconate (0.0 to 1.0%), and N-acetylglucosamine (0.0 to 1.0%) were added to skim milk to retain the viability of Lactobacillus acidophilus and Bifidobacterium longum. To carry out response surface modeling, the regression method was performed on experimental results to build mathematical models. The models were then formulated as an objective function in an optimization problem that was consequently optimized using a genetic algorithm approach to obtain the maximum viability of the probiotics. The genetic algorithms (GAs) were examined to search for the optimal value. The results indicated that GAs were very effective for optimizing the activity of probiotic cultures.