Lactobacillus fermentum (strain RS2) is a common resident of the human gastrointestinal tract (GIT) and used as potential probiotics. The ability to adapt, colonize and survive in the harsh environment of the GIT, in the presence of gastric juice, is one of the unique properties that helps microflora to transiently harbor the host. In the current study, we used gel free quantification approach coupled with a high-resolution mass spectrometer to analyze the expression pattern of Lactobacillus fermentum RS2 strain under 1.2% bile acid stress condition. The analysis resulted in the identification of 1157 varieties of proteins that are engaged in coping up the efflux of bile effect or protons, by altering the carbohydrate metabolic pathways or through prevention of protein misfolding. The function of downregulated proteins were mostly unknown and the putative functions of the upregulated proteins were categorized as stress response, DNA repair, amino acids metabolism, signal transduction, transcription, translation, and carbohydrate metabolism. The finding suggests that these proteins are involved in bile resistance specific mechanisms as well as processes responsible for adaptation in GIT and showed the association of expression levels of proteins with a precise probiotic trait.