We report the first analysis of the soluble sub-proteome of the obligate thermophile, Geobacillus thermoleovorans T80, utilizing a robust multidimensional protein identification protocol. A total of 1,336 proteins were initially identified utilizing automated MS/MS identification software. Intensive manual curation resulted in a final list containing a total of 294 unique proteins. Physiochemical characterization and functional classification of the soluble sub-proteome was carried out. The strategy has allowed us to gain an insight into the cellular processes of this obligate thermophile, identifying a variety of proteins known to play a role in stress response. Included within these were a number of sigma factors such as sigma(A) that initiate transcription of the heat shock operons controlled by the HrcA-CIRCE complex within gram positive bacteria. In addition, it has enabled us to assign a degree of functionality to 29 out of 36 gene products detected in this study that were hitherto described as being only hypothetical conserved proteins.