Quantification of fumaric acid, an endogenous dicarboxylic acid with interesting biomedical applications either through its own biological activity or as a linker constitutive of the porous iron(III) fumarate metal organic framework (MOF) MIL-88A based drug nanocarrier (MIL stands for Material from Institut Lavoisier), has been developed in different rat biological complex media (liver, spleen and urine). After a liquid–liquid extraction procedure, fumaric acid concentration was determined by a simple and accurate high-performance liquid chromatography (HPLC) method coupled to a photodiode-array detector (PDA) using aminosalicylic acid as internal standard (IS) and a gradient elution. The recovery of fumaric acid reaches 89% and 92% for urine (for concentrations of 0.05 and 1 μg ml−1, respectively) and 90% for liver and spleen tissues, exceeding 89% in all instances in comparison with the IS. Linearity has been kept from 0.05 to 1 μg ml−1 and from 0.5 to 10 μg g−1 of fumaric acid in urine and tissues, respectively. The limit of detection of the method was 0.01 μg per injection. This method has finally allowed the quantification of fumaric acid in rat urine and tissue samples after the intravenous administration of MIL-88A nanoparticles.