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A new method has been developed for the spectrophotometric determination of p-aminophenol (PAP) in water, paracetamol formulations and human urine samples with a recovery rate between 94.9 and 101%. This method exploits an aqueous two-phase system (ATPS) liquid-liquid extraction technique with the reaction of PAP, sodium nitroprusside and hydroxylamine hydrochloride in pH 12.0, which produces the [Fe(2)(CN)(10)](10-) anion complex that spontaneously concentrates in the top phase of the ATPS ([Formula in text]). The ATPS does not require an organic solvent, which is a safer and cleaner liquid-liquid extraction technique for the determination of PAP. The linear range of detection was from 5.00 to 500 μg kg(-1) (R ≥ 0.9990; n=8) with a coefficient of variation of 2.11% (n=5). The method exhibited a detection limit of 2.40 μg kg(-1) and a quantification limit of 8.00 μg kg(-1). The ATPS method showed a recovery that ranged between 96.4 and 103% for the determination of PAP in natural water and wastewater samples, which was in excellent agreement with the results of the standard 4-aminoantipyrine method that was performed on the same samples., (Copyright © 2011 Elsevier B.V. All rights reserved.)