The DFO, a special hexadentate chelator with three hydroxamate moieties, is a bifunctional para-thiocyanatobenzyl-decafluorobenzoate (p-SCN-Bn-Df), a significant next-generation ligand. The presence of the thiocyanate (-SCN) group, which is capable of hydrolysis and protonation processes. The aim of this study is to determine the stability of 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(n-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosine] thiourea (p-SCN-Bn-Df) via RP-HPLC method. A variety of mobile phases were tested in various ratios of methanol: water, acetonitrile: water and phosphate buffer at various pH levels. However, when employing a mobile phase consisting of water to acetonitrile containing 0.1% TFA (05:95, v/v) in an isocratic manner, satisfactory separation and symmetric peaks were observed. In this method utilized Eclipsed C-18 column (5μm, 4.6 × 250 mm) with a flow rate 0.5 mL/min. The maximum absorption wavelength of p-SCN-Bn-Df at 254 nm was selected as detection wavelength. The retention time (t_R) of p-SCN-Bn-Df was found at 5.205 min. The ICH guideline was used to evaluate linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), specificity and system appropriateness criteria to validate the optimized chromatographic and spectrophotometric procedures. For accurate compound separation in pharmaceutical and environmental analyses, this phase is adaptable and often used. This study is useful of p-SCN-Bn-Df for evaluating QC parameters and chelation rates with different radioisotopes e.g. Zr-89.