Probiotic bacteria are live microorganism which confer health benefits in humans. Several types of probiotics are in use and to explore the new strains, the safety issues have to be addressed. Assessment should be considered at level of type of strain, physiological action, mode of administration, dosage, and the health condition of users. However, one of the most important concerns is the translocation of administered probiotics into bloodstream and internal organs through intestinal barrier. In this chapter, we have explained the method for the in vivo assessment of probiotic translocation into bloodstream and internal organs.