For the first time the phytosiderophore 2'-deoyxymugineic acid (DMA) could be accurately quantified by LC-MS/MS in plant and soil related samples. For this purpose a novel chromatographic method employing porous graphitic carbon as stationary phase combined with ESI-MS/MS detection in multiple reaction monitoring was developed. Isotope dilution was implemented by using in-house synthesized DMA as external calibrant and (13) C4 -labeled DMA as internal standard (concentration levels of standards 0.1-80 μM, determination coefficient of linear regression r(2) > 0.9995). Sample preparation involved acidification of the samples in order to obtain complete dissociation of metal-DMA complexes. Excellent matrix related limits of detection and quantitation depending on different experimental setups were obtained in the range of 3-34 nM and 11-113 nM, respectively. Standard addition experiments and the implementation of the internal (13) C4 DMA standard proved the accuracy of the quantification strategy even in complex matrices such as soil solution. The repeatability of the method, including sample preparation, expressed as short- and long term precision was below 4% and 5% RSD, respectively. Finally, application in the context of plant and soil research to samples from rhizosphere sampling via micro suction cups, from soil solutions and soil adsorption/extraction studies revealed a DMA concentration range from 0.1 to 235 μM. This article is protected by copyright. All rights reserved.