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Performance and efficiency of feed utilization in poultry is highly influenced by gut health, which is dependent on intestinal microbial balance. Probiotics are live microbial feed supplements or viable microorganisms that beneficially affect the host animal by improving its gastrointestinal tract (GIT) microbial balance. However, their mode of action and suitable GIT environment favoring their colonization of the GIT is obscure. The probiotic properties of Lactobacillus plantarum, Bifidobacterium longum, and Saccharomyces boulardii were evaluated. These microbes were tested in vitro against gastrointestinal conditions for survivability and their ability to attach to the intestinal mucosa. The ability of the microbes to tolerate and survive varying pH levels and bile concentrations was assessed. The microbes were challenged with a pH of 2 to 7 for 5 h and bile concentrations of 1 to 3% for 6 hrs. The microbes were sampled hourly to evaluate growth or decline in colony-forming units (CFU). B. longum, L. Plantarum, and S. boulardii exhibited significantly higher CFU (p < 0.05) at a pH range of 5 to 7, 4 to 7, and 2 to 7, respectively, when compared with other pH levels. L. plantarum had much higher colony-forming units per mL within each pH level, except at pH 2 where S. boulardii was the only microbe to survive over time. While L. plantarum and S. boulardii were able to tolerate the various bile concentrations, B. longum and L. plantarum showed remarkable ability to attach to the intestinal mucosa and to inhibit pathogenic microbes.