Several iron binding metabolites (siderophores) of Pseudomonas fluorescens B10 (JL-3133) have been detected using C18 reverse phase HPLC coupled with photodiode array detection methods. This analysis utilized a volatile mobile phase of 90% 20 mM NH4HCO3/10% MeOH, pH 6.5. It has been shown to be applicable to other P. fluorescens strains for the detection of related metabolites. Direct scale-up of the analytical HPLC conditions allowed for the efficient preparative isolation of pseudobactin, the principle siderophore produced by P.fluorescens B10 (JL-3133).