The use of bacterial strains was found in some articles the activity of Levofioxacin. However, technical details about the methodology and the validation of hioassay method were not described. Rodriguez et al. [271 evaluated the in vitro activity of Levofloxacin against different strains of M mberculong. Similarly the concentration of Levofloxacin in ophthalmic solution was simple measured using B. rubrilis ATCC-6633 261. In the proposed study eutical 16 suains of bacteria were tested for their response and susceptibility against Levofloxacin. Although most of the tested strains showed susceptibility against Levofloxacin. B. pumiks ATCC 14884 was selected as the most significant microbial suaan iecause of its high response and capacity to form sharply defined zone of inhibition To quantify antibiotie through microbial bioassay, the inoculum concentration should be validated. which showed sharT and clear antibiotic zone of inhibition 1311. An influence of inoculum concentrtion on resulting zone size is widely recog nized and experiments were performed to determine how critical