A simple, rapid and reproductible method by extraction-derivative spectrophotometry, without the classic deproteination step, is proposed for the direct determination of serum iron, This method is based on the integration of liquid-liquid separation and reaction at pH = 5.0 of iron with (4-phenyl-2-pyridinyl)-5-phenyl-1,2,4 triazine (PPT) and 2,4,6-trinitro-phenol (picric acid) in dichloroethane. The reaction and the extraction are quantitative, being the proteins free of iron. A phase separator (Whatman IFS) was also used, which permits the separation of organic phase containing the analyte, as the iron(II)-PPT-picrate complex. The proteins were retained quantitatively. The iron content were measured by first-derivative absorption spectrum, using Delta lambda = 20 nm. As the method is free from cation interferences it was successfully applied to the determination of iron in human serum.