Reasons for the selected range of concentration were the susceptibility of microorganism to low concentrition, size of the zones of inhibition that was limited by the size of Petri dish for high concentration and linear relationship between the logarithm of concentration and mean zane diameters. The calibration value of Levofloxacin was constructed by plotting the logarithm of antibiotic concentration(ugimL) versus mean dia- meter of inhibition zone(in mm) and good linearity was found in the range of the selected conceauations of the reference standard. A comparative study of microbiological bioassay was also camed out with the HPLC method and the potency of Levoflox- acin was estimated as 100.90% and 99.37%. respectively. The equivalence in results shows a strong comelation between these two methods. However, estimating the bioactivity merobiological assay is an effective method of determining the subtle change in the antibiotic.