The aim of this study was to compare the survival of chosen isolates of LAB in artificial duodenal fluid contained the cholesterol (0.5 g/L) with the survival in control duodenal fluid without the cholesterol addition. Isolates of five species of LAB (bifidobacteria, Lactobacillus acidophilus, Lb. delbrueckii subsp. bulgaricus, Lb. plantarum, Lb. rhamnosus) isolated from commercial pharmaceuticals and isolates of six species of LAB (bifidobacteria, Lb. acidophilus, Lb. delbrueckii subsp. bulgaricus, Lb. casei, Lactococcus lactis, Streptococcus thermophilus) originated from commercial dairy products or dairy starter cultures were used in the study. Duodenal fluid contained 5.0 g/L of NaCl, 0.6 g/L of KCl, 0.03 g/L of CaCl₂, 17 g/L of bile salts, and the enzyme complex (pancreatin enzymes, lipases, amylases, and proteases) dissolved in 1 M NaHCO₃. The pH of the juice was 7.0 ±0.2. The survival rate of studied LAB in simulated duodenal fluid did not depend on the cholesterol addition. The LAB Isolates originated from pharmaceuticals survived in simulated duodenal fluid as well as the isolates originated from dairy starter cultures or dairy products. The survival rate of studied LAB in simulated duodenal fluid depended on the initial count of bacteria. The highest initial number of LAB the highest number of bacterial cells survived in simulated duodenal fluid.