Mycobacterium acquire iron by producing siderophores called mycobactins, with an extremely high affinity complexation of Fe3+. The iron complex shows distinctive electrochemistry predicting 18 orders of magnitude greater affinity of the mycobactin for Fe3+ than Fe2+. A heterogeneous standard rate constant, ks of the order of 10−5 cm s−1 confirms quasi reversible electrochemistry and based on the equilibrium in the presence of excess solution Fe3+/Fe2+ the oxidation and reduction peaks for the siderophore complex could be calibrated for ferric mycobactin J (FeMJ). FeMJ spiked urine collected from cows showed a matrix effect on the current peak height. For example, 240±15 µM FeMJ was estimated in 600 µM spiked urine. However, in the presence of excess solution Fe(acac)3 the same sample yielded an estimated 580±25 µM FeMJ.This study was supported by the Tuberculosis ResearchSection of Clifton E. Barry III as part of the Intramural Research Program of the National Institute of Allergy andInfectious Diseases, U.S. National Institutes of Health in the form of a stipend for author NSM.This is the accepted manuscript of a paper published in Electroanalysis Special Issue: Mátrafüred 2014 – International Conference on Electrochemical Sensors Volume 27, Issue 3, pages 833–842, March 2015, DOI: 10.1002/elan.20140056