Bacillus atrophaeus and Bacillus pumilus spores are widely used as biological indicators to assess the effectiveness of decontamination procedures. Spores are intricate, multi-layered cellular structures primarily composed of proteins, which significantly contribute to their extreme resistance. Therefore, conducting a comprehensive proteome analysis of spores is crucial to identify the specific proteins conferring spore resistance. Here, we employed a high-throughput shotgun proteomic approach to compare the spore proteomes of B. atrophaeus DSM675 and B. pumilus DSM492, identifying 1312 and 1264 proteins, respectively. While the overall number of proteins found in both strains is roughly equivalent, a closer examination of a subset of 54 spore-specific proteins revealed noteworthy distinctions. Among these 54 proteins, 23 were exclusively detected in one strain, while others were shared between both. Notably, of the 31 proteins detected in both strains, 10 exhibited differential abundance levels, including key coat layer morphogenetic proteins. The exploration of these 54 proteins, considering their presence, absence, and differential abundance, provides a unique molecular signature that may elucidate the differences in sensitivity/resistance profiles between the two strains.