While lactic acid bacteria and bifidobacteria have been scientifically important for over a century, many of these are marketed today as probiotics and have become a valuable and rapidly expanding sector of the food market that is leading functional foods in many countries. The human gastro-intestinal tract with its various compartments and complex microbiota is the primary target of most of these functional foods containing lactic acid bacteria and bifidobacteria (LAB&B). In addition, their use as vectors for delivery of molecules with therapeutic value to the host via the intestinal tract is being studied. This review focuses on molecular approaches for the investigation of the diversity of lactic acid bacteria and bifidobacteria in the human intestine, as well as tracking of probiotic bacteria within this complex ecosystem. Moreover, methodologies to determine the viability of the lactic acid bacteria and bifidobacteria and molecular approaches to study the mechanisms by which they adapt, establish and interact with the human host via the digestive tract, are described.