Several proteins have been identified in the past decades as targets of uranyl (UO$_2$$^{2+}$) in vivo. However, the molecular interactions responsible for this affinity are still poorly known which requires the identification of the UO$_2$$^{2+}$ coordination sites in these proteins. Biomimetic peptides are efficient chemical tools to characterize the nature of these sites. The aim of this work is to develop a dedicated analytical method to determine the affinity of biomimetic synthetic multi-phosphorylated peptides for UO$_2$$^{2+}$ and to evaluate the effect of several structural parameters of these peptides on this affinity at physiological pH. The analytical strategy was based on the implementation of the simultaneous coupling of hydrophilic interaction chromatography (HILIC) with electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). An essential step had been devoted to the definition of the best separation conditions of UO$_2$$^{2+}$ complexes formed with di-phosphorylated peptide isomers and also with peptides of different structure and degrees of phosphorylation. We developed in this work the first separations of several sets of UO$_2$$^{2+}$ complexes by HILIC ever reported in the literature. A dedicated method had then been developed for identifying the separated peptide complexes online by ESI-MS and simultaneously quantifying them by ICP-MS, based on uranium quantification using external calibration. Thus, the affinity of the peptides for UO$_2$$^{2+}$ was determined and made it possible to demonstrate that (i) the increasing number of pSer promotes the affinity of the peptides for UO22+, (ii) the position of phosphorylated residues (pSer) in the peptide backbone has very low impact on this affinity (iii) and finally the cyclic structure of the peptide favors the UO$_2$$^{2+}$ complexation in comparison with the linear structure. The results obtained using this approach are essential to better understand the mechanisms of toxicity of UO$_2$$^{2+}$ at the molecular level and to further develop selective decorporating agents by chelation