In recent years the roles of probiotics as functional ingredients in food has been highly adopted by the consumers and are under constant investigation by the scientific community. As a result, several probiotic-containing foods have been introduced in the market with an annual share of several billion dollars. Of particular interest in the probiotics research is the profiling of probiotic character of the microbes involving both in vitro and in vivo approaches. Initially traditional microbiological techniques were used; however they suffer by many limitations and therefore the development of new techniques, which are primarily based on the analysis of nucleic acids have been introduced. The scope of this review is to present current knowledge about the methodological approaches that are used to quantify and characterize the potential probiotic character of microorganisms. Moreover, it will focus on molecular and non-molecular tools and finally will report some new perspectives in the study of probiotics using omics techniques.