Mammalian histidine-rich glycoprotein (HRG) is a highly versatile and abundant blood plasma glycoprotein with a diverse range of ligands that is involved in regulating many essential biological processes, including coagulation, cell adhesion and angiogenesis. Despite its biomedical importance, structural information on the multi-domain protein is sparse, not least due to intrinsically disordered regions that elude high-resolution structural characterisation. Binding of divalent metal ions, particularly ZnII, to multiple sites within the HRG protein is of critical functional importance and exerts a regulatory role. However, characterisation of the ZnII binding sites of HRG is a challenge; their number and composition, as well as their affinities and stoichiometries of binding are currently not fully understood. In this study, we explored modern electron paramagnetic resonance (EPR) spectroscopy methods in combination with AlphaFold2 protein structure prediction to assemble a holistic picture of native HRG and its interaction with metal ions. To our best knowledge this is the first time this suite of EPR techniques has been applied to count and characterise endogenous metal ion binding sites in a native mammalian protein of unknown structure.