Iron is an essential element in human development. It is imperative for oxygen and electron transport and also for DNA and neurotransmitters synthesis. On the other hand, this metal is able to participate in Fenton's reaction that in turn leads to free radical damage. The most toxic fraction of iron - nontransferrin-bound iron and its part desferrioxamine-chelatable iron - can serve as an exquisite biomarker in the identification of iron imbalance. The goal of the present study was to devise a simple, repeatable, and inexpensive method for the determination of desferrioxamine-chelatable iron in serum blood samples. The assay procedure is based on desferrioxamine complex formation with iron ions followed to ferrioxamine and its quantitative measurement using RP-HPLC method. The desferrioxamine-chelatable iron was extracted from blood by centrifugation and SPE method. Chromatographic separation was performed at 40°C by step-form gradient elution using Cadenza CD-C18 column (150 × 4.6 mm id, particle size of 3.0 μm) connected with precolumn for contaminants removal. Gradient HPLC elution has been carried out with solvent A (10 mM Tris-HCl, pH 5.5) and solvent B (ACN). The flow rate was 1.2 mL/min, and the total separation time was 5 min. The linear quantitation range was 2.5-500 μM (r = 0.9973), and the LOD and LOQ were 0.42 and 1.29 μM, respectively. Proposed HPLC method allowed for the determination of desferrioxamine-chelatable iron fraction's of nontransferrin-bound iron, both in the buffer and the serum supplemented with iron ions as well as in the patients' serum samples with good results of precision and recovery. The developed method found to be sufficiently precise and reproducible for established conditions and after validation and may be used for routine assay of desferrioxamine-chelatable iron in biological samples.