Adhesion of probiotic bacteria to intestinal epithelial cells is regarded as a prerequisite to exert beneficial health effects. Human intestinal epithelial lines, like Caco-2 or HT-29 cells, have been extensively used to select for adhesive strains in vitro. Adhesion to intestinal mucus has been used to a lesser extent. However, to date, there has been no standardization of the conditions used in in vitro adhesion assays. As a consequence, results obtained in different laboratories using identical strains, but different assay conditions, show great variability. This lack of standardization complicates the interpretation of data and discrepancies emerge on what is meant by effective adhesion. A critical validation of in vitro adhesion is essential for the food industry, using adhesion assays as predicitve screening tools to assess new probiotic strains. In this review we summarize a comparative study on adhesion of the well characterized probiotic bacteria L. johnsonii La1 and L. rhamnosus GG with respect to the influence of bacterial growth conditions, growth phase, buffer, pH, and mucus on adhesion properties. The results are employed in the current scientific discussion to allow a critical evaluation on the reliability of the in vitro assays.