Bacillus clausii, a gram positive, spore forming bacteria is widely used a probiotic in preventing and treating various gastrointestinal disorders. In order to understand the functioning of this strain as a probiotic, one needs to study the molecular level of interaction of this strain with host systems. Considering the fact that the first level of interaction can happen at the extracellular level, the project aims to characterize extracellular matrix proteins of Bacillus calusii. The workflow includes extraction, isolation and characterization of the extracellular proteins from Bacillus calusii using various analytical tools such as affinity chromatography, PAGE and mass spectrometry (MS). The MS based proteomic analysis revealed five surface proteins viz. UVRABC system protein, alkaline phosphatise, putative sugar-lactose lactonase, uncharacterized YRVC and endospore coat associated proteins. These proteins are thus important targets for elucidating the molecular cross talk between Bacillus clausii and their hosts.