Streptomycin reacts with ninhydrin in alkaline medium to give a violet colour having maximum absorbance at 560 nm. The reaction is specific for streptomycin with 0.1 μg/ml as minimum limit of detection. It also provides the basis for a new spectrophotometric determination of pure streptomycin and in the presence of the organic compounds. The maximum tolerable limits of these are reported. The method is successfully applied to the determination of streptomycin from pharmaceutical preparation.