Phytosiderophores (PS) form stable complexes with various transition metals. These ligands are exuded by the roots of graminacous plants as a mechanism for mobilizing and acquiring soil iron. To investigate iron mobilization and transport, a novel LC method in combination with ESI-MS/MS for the determination of three Fe(III)-complexes with mugineic acid (MA), 2'-epi-MA and 2'-deoxymugineic acid (DMA) has been developed. Liquid chromatographic separation was realized using a silica-based mixed-mode reversed phase/weak-anion exchange type stationary phase and a 50 mM ammonium acetate buffer, pH 6.5. Baseline separation of the two complex diastereomers Fe(III)-MA and Fe(III)-epi-MA could be achieved. ESI-MS/MS detection allowed for simultaneous quantification of the complexes and the free ligands. Limits of detection were determined to be 0.001 and 0.05 mu M for DMA and Fe(III)-DMA, respectively. The analytical figures of merit of the novel method were evaluated and compared with a CE-ESI-MS method that we had published earlier. The LC-ESI-MS/MS method has been successfully applied to real samples derived from preliminary extraction experiments.