Intake of probiotics is associated with many health benefits, which has generated an interest in formulating viable probiotic supplements. The present study had two aims. The first aim was to achieve gastrointestinal protection and delayed release of viable probiotics by pelletizing and coating freeze-dried probiotic strains, using riboflavin as a marker for release. The second aim was to set up a dynamic three-step in vitro model simulating the conditions in the human gastric, duodenum/jejunum and ileum compartments using physiologically relevant media to evaluate delayed release of the formulations. To simulate lowered bile acid concentrations in the ileum area of the gastrointestinal tract, a novel method using the bile acid sequestrant cholestyramine to lower bile acid concentrations in the small intestinal medium to physiologically relevant levels was attempted. Granulation, extrusion and spheronization was used to develop pellets containing viable probiotics using freeze-dried Lactobacullus reuteri as a model strain. Fluid bed coating the pellets with the pH-sensitive polymers Eudragit S100 or Eudragit FS30D resulted in targeted release in the ileum step of the three-step in vitro model based on release of the marker riboflavin.