Propionibacterium freudenreichii is the representative dairy propionic bacteria, best known for its long tradition of use in the production of Swiss-type cheeses. Today its importance extends beyond cheese-making due to a high capacity for vitamin B12 biosynthesis and a growing potential for application in probiotic food products. Employing proteomics in the study of this bacterium aims to provide an in-depth understanding of its physiology as well as valuable tools for future application-orientated strain selection. However, the extent of proteomic research into P. freudenreichii is to date very limited. This work describes the two-dimensional gel electrophoresis (2-DE) - based comparison of two strains of P. freudenreichii, as well as the optimization of the required proteomic protocols. It should prove useful to students of Life Sciences and Food Sciences and researchers with an interest in gel-based bacterial proteomics and the study of potential probiotics.