In the bio-mining industry there are various compounds which need to be frequently monitored, one of which is iron, since its concentration can seriously affect the industrial process. This study describes the development of an electrochemical method of speciation and rapid quantification of iron in bio-mining processes conditions. Speciation and quantification was carried out in a flow injection system using techniques of cyclic voltammetry and chronoamperometry. The proposed and a standard method were used to compare the results obtained from simulated and real mixed (ferric/ferrous) samples. Cyclic voltammograms presented two peaks corresponding to the ferric ion reduction of and ferrous ion oxidation. Chronoamperometry allow the construction of calibration curves for both ions. Quantification of real and simulated samples showed acceptable levels of error compared to a standard method. The electrochemical method allowed in a simple and rapid way the speciation and quantification of iron in mixed samples, including real samples from acid mine drainage, which established the basis for the development of an automated process monitoring device.