Aims: To develop an easy-to-use and pathogen-free protocol giving reliable information on the bioavailability of iron in a medium. Methods and Results: In aerobic conditions, iron bioavailability is very low, and most of its forms cannot be assimilated by micro-organisms. Media with similar iron contents can differ considerably in iron bioavailability, something that is not easily achieved using conventional physicochemical methods. The assay developed in the present work is based on a pyoverdin siderophore release by fluorescent Pseudomonas in response to iron stress. Conclusions: The test was applied to a complex medium used for the production of diphtheria toxin (DT). A significant difference between the bioavailable iron level and the total chemical concentrations contributed by the various compounds used to make the medium could thus be detected. This can be explained by the formation of salt complexes trapping the iron, which thus cannot be used directly by the micro-organism for its metabolism. Significance and Impact of the Study: The assay can easily be applied to any medium designed for the production of iron-regulated compounds. This is particularly useful when dealing with processes that use pathogenic strains as was shown in the case based on DT production.