Probiotic formulations are often used in prophylaxis and as a supportive therapy in the case of gastrointestinal tract function disorders in adults and infants. Numerous monostrain or multistrain probiotic formulations are available on the market. Before launching of the probiotic product, the mechanisms of action must be scientifically proven and described. The aim of this work was to validate the quality of a randomly selected, commercially available formulation for infants, by assessing the number of probiotic cells and content strains, and by analyzing the survival of strains in simulated conditions of the gastrointestinal tract. According to that, microbiological analyses were applied, and the analysis of the present probiotic cultures was carried out using the next generation sequencing approach. Following the results, formulation contains the number of bacterial cells as stated on the declaration and exceeds the value of the minimum prescribed concentration of bacterial cells per milliliter of probiotic formulation, which fulfills the key requirement for assessing the quality of probiotics. The probiotic consortium effectively survived simulated conditions of the gastrointestinal tract. Sequencing analysis confirmed the presence of Lactobacillus rhamnosus GG, Lactobacillus acidophilus and Lactobacillus reuteri strains. The presence of contaminating bacteria is excluded. The results of this work confirm the importance of continuous monitoring of the quality of probiotic formulations, especially those produced according to the principles of good manufacturing practice, with the aim of reducing the number of unreliable formulations and ensuring the use of high-quality probiotic products.