Ceftiofur Sodium is a semi-synthetic beta-lactamase broad spectrum third generation of cephalosporin antibiotic. The development and validation of a two-level agar diffusion (2+2) bioassay to quantify ceftiofur in powder for injection is described in this paper. Bacterial strain Bacillus subtilis ATCC 6633 (MTCC 441) was selected and used as the most significant strain against ceftiofur sodium. The mean potency recovery value of ceftiofur sodium in marketed sample XCEFT powder for injection was estimated to be 101.79%. All potency results were statistically analyzed and found to be linear (r2 = 0.9916) in the range of 1.0-10 µgmL-1, with the intermediate precision RSD between days was 0.81%; intermediate precision RSD between analyst was 0.43% and accuracy 101.08%, RSD = 0.28%. The findings backed up the proposed microbiological technique, which allowed for accurate ceftiofur sodium quantification in pharmaceutical samples. Furthermore, bioassay is a useful, easy and cost-effective method for controlling the quality of ceftiofur sodium in raw material as well as in pharmaceutical preparations.