A new micelle-mediated phase separation method using an anionic surfactant, sodium dodecyl sulfate (SDS), for preconcentration and spectrophotometric determination of Pb(II) and Cu(II) is presented. The procedure is based on the complex-formation reaction between Pb(II) / Cu(II) and a synthesized chelating agent, N1,N2-diphenylhydrazine-1,2- dicarbothioamide (PHCT) and subsequent extraction of the colored complex using sodium dodecyl sulfate which was used as extracting agent at room temperature. The chemical variables affecting the separation and determination procedure were optimized. In optimum conditions Beer’s law was obeyed in the range 0.01–0.90 µg mL-1 for both cations. Limit of detection at the wavelength of the maximum absorption was found to be 2 ng mL-1 and 3 ng mL-1 for Pb(II) and Cu(II), respectively. The proposed method was applied successfully to the determination Pb(II) and Cu(II) in natural and waste water, and spiked human plasma samples.