The present study reports an in-depth proteome analysis of two Lactobacillus rhamnosus strains, the well-known probiotic strain GG and the dairy strain Lc705. We used GeLC-MS/MS, in which proteins are separated using 1-DE and identified using nanoLC-MS/MS, to generate high-quality protein catalogs. To maximize the number of identifications, all data sets were searched against the target databases using two search engines, Mascot and Paragon. As a result, over 1600 high-confidence protein identifications, covering nearly 60% of the predicted proteomes, were obtained from each strain. This approach enabled identification of more than 40% of all predicted surfome proteins, including a high number of lipoproteins, integral membrane proteins, peptidoglycan associated proteins, and proteins predicted to be released into the extracellular environment. A comparison of both data sets revealed the expression of more than 90 proteins in GG and 150 in Lc705, which lack evolutionary counterparts in the other strain. Differences were noted in proteins with a likely role in biofilm formation, phage-related functions, reshaping the bacterial cell wall, and immunomodulation. The present study provides the most comprehensive catalog of the Lactobacillus proteins to date and holds great promise for the discovery of novel probiotic effector molecules.