The popularity of the probiotics in India has expanded exponentially as they facilitate the host animals in improving the intestinal imbalance of microbial flora. They can be administered as pellets, capsules, paste, powder, curd, granules and also in form of suspensions. In some countries probiotics are also taken as prophylactic agents. Bacillus clausii spore suspension is a probiotic of choice in the treatment of diarrhoea and prevention of antibiotic associated diarrhoea. The spores have catapulted themselves as the prescription choice for the treatment of diarrhoea in India. In the present study, three commercially available Bacillus clausii spore suspensions were collected for the phenotypic characterization and biochemical analysis. The optimal viability assay conditions and the antibiotic susceptibility assay profiles are determined. The optimization of the key parameters is also presented. Sequence alignment was performed to check the evolutionary relationship of Bacillus clausii with organisms of different taxa. Statistical study was performed by using BEAST and a tree was constructed.