Rhizopus arrhizus was grown in an iron-free nutrient solution growth medium. Mass production of the siderophores was achieved in a short time by continuous growth of the fungi: after the secretion of the siderophores into the growth medium the spent medium was collected and replaced by a fresh medium while the fungus mat was kept in the flask. The medium exchange was repeated several times and the optimal time for the collection of the siderophore was found to be when the fungus was fully developed, usually about 3 days after the exchange. It was found that it is feasible to grow the fungi continuously for five growth periods, after which the fungus culture becomes too old and collapses. The siderophore was isolated and cleaned from the spent medium using a series of columns on an FPLC at room temperature. Additional tests were used to verify the existence of the siderophore in the solution. The concentrations of rhizoferrin in the various fractions was measured using an exchange reaction between the siderophore and that of an added chelate solution (CAS) while employing a series of dilutions of the CAS. For a precise analytical determination of the siderophore rhizoferrin, Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICR-MS) was used to validated that the siderophore is indeed rhizoferrin which has been structurally identified earlier. © 2017 by Koninklijke Brill NV, Leiden, The Netherlands.