Simultaneous and fractional determination of iron(II) and iron(III) was accomplished with o-hydroxyhydroquinonephthalein (QP) in the presence of poly (N-vinyl pyrrolidone). In the determination of total iron (iron(II)+iron(III)), Beer's law was obeyed in the range of 0.02-0.67 μg·ml⁻¹, with an effective molar absorptivity (at 570 nm) and a relative standard deviation of 1.30×10⁵·l·mol⁻¹·cm⁻¹ and 0.77% (n = 8), respectively. This method was about 10-15 times and more than the methods using 1,10-phenanthroline and 2,2′-bipyridine. In addition, the iron-QP complex was characterized using spectrophotometry and the electron spin resonance. This method was successfully applied to assays of total iron and iron(III) in pharmaceutical preparations.