In thispaper,theuseof3-hydroxy-4-pyridinone(3,4-HPO)chelatorsasnontoxicchromogenicreagents for irondeterminationisproposed.Thepotentialapplicationofthesecompoundswasstudiedina sequentialinjectionsystem.The3,4-HPOligandsusedinthisworkwerespeciallydesignedtocomplex iron(III) atphysiologicpHforclinicalapplications.Thedevelopedsequentialinjectionmethodenabled to studythereactionconditions,suchasbufferingandinterferences.Then,tofurtherimprovethelow consumptionlevels,amicrosequentialinjectionmethodwasdevelopedandeffectivelyappliedtoiron determinationinbathingwatersusing3,4-HPOligands.Theformedironcomplexhasamaximum absorbanceat460nm.Theadvantageofusingminimalconsumptionvaluesassociatedwithsequential injection,togetherwiththelackoftoxicityof3,4-HPOligands,enabledtopresentagreenerchemistry approachforirondeterminationinenvironmentalsampleswithintherange0.10-2.00mgFe/Lwitha LOD of7 mg/L. Theoveralleffluentproductionwas350 mL correspondingtotheconsumptionof0.48mg of 3,4-HPOligand,0.11mgofNaHCO3, 0.16mgofHNO3 and 50 mL ofsample.Threereferencesamples were assessedforaccuracystudiesandarelativedeviation o5% wasobtained.Theresultsobtainedfor the assessmentofironininlandbathingwaterswerestatisticallycomparabletothoseobtainedbythe referenceprocedure.