A new, simple, sensitive and selective spectrophotometric method for the determination of streptomycin (SM) using o-hydroxyhydroquinonephthalein (Qn. Ph.) and manganese (II) [Mn (II)] in 40% methanolic media was established. This method is based on the fact that the SM-Mn (II)-Qn. Ph. complex shows a red shift in weakly basic media as compared with the Mn (II)-Qn. Ph. complex. This method could be used to determine up to ∼25 μg/10 ml of SM ; the apparent molar absorptivity was estimated to be 2.4×10⁵ l mol^-1cm^-1 at 570 nm. Guanidino compounds appear to be involved in this color reaction. Recovery of SM added to human urine was good.