Bifidobacteria are gram-positive anaerobes with a high Guanine/Cytosine genome content. Specific strains are used as probiotics because of their health benefits. Probiotics are live microorganisms with a positive influence on their host's health. They are used as nutritional supplements and their resistance to conditions of food manufacturing and of the gastro-intestinal tract is studied. We report on differential proteomics of two Bifidobacterium longum strains that differ in their heat shock resistance. This was achieved by a comparative qualitative survey of their proteomes and relative LC-MS/MS-based label-free protein quantification. Deploying a nano LC-ESI ion-trap mass spectrometer, 165 proteins expressed by the two probiotic strains were identified. Around 50% of these were common to both strains with the remaining half identified in either one of the strains. Using a label-free technology based on the 3D overlay of retention times, mass-over-charge ratios and ion intensities between LC-MS runs, we found quantitative differences in the relative abundance of 19 of the proteins common to both strains. The differentially expressed proteins were classified into categories involving glucose metabolism, protein synthesis and heat shock proteins. Six of these 19 proteins have been previously reported as being associated to heat stress response.