Background: Commercially prepared food products containing sucralose are becoming increasingly popular among consumers. Aims and Objectives: This study determined the effects of varying concentrations of sucralose on the acidproducing capability of Lactobacillus acidophilus. Materials and Methods: L. acidophilus was inoculated in de Man, Rogosa, and Sharpe broth selective medium containing varying concentrations of sucralose per kg broth powder. The change in pH was measured after 48 h of culture using a pH meter. The data were analyzed using one-way analysis of variance and Bonferroni pairwise comparison test at 5% level of significance. Results: The largest mean pH change of 0.120 was observed in 250 mg/kg, followed by 0.070 in the 350 mg/kg treatment group. The control and 500 mg/kg treatment groups had the least observed mean pH changes of −0.010 and −0.008, respectively. These mean pH changes varied significantly among all treatment groups. Conclusion: There was a decrease in the change in pH as the concentration of sucralose was increased suggesting that the addition of certain concentrations of sucralose influenced the acid production of L. acidophilus.