A simple, sensitive and specific agar cup diffusion bioassay for the antibacterial Colistin sulfate was developed. Using a strain of Escherichia coli ATCC 8739 as the test organism, Colistin sulfate at concentrations ranging from 100 to 1600 µg/ml could be measured in pharmaceuticals. A prospective validation of the method showed that the method was linear (r 2 = 0.999), precise (RSD< 2.8%) and accurate (percent recovery ranges between 98-102%). The method shows that results confirm its precision, not differing significantly from the other method described in the literature. We conclude that microbiological assay is satisfactory using Escherichia coli ATCC 8739 for quantitation of in-vitro antibacterial activity of Colistin sulfate.