Hydroxyapatite ((Ca10(PO4)6(OH)2, HAp) based chromatography matrix has attracted great interest in the field of protein separation. However, researchers have been trying to combat the growing costs associated with the HAp matrix. In the present investigation, we utilized a cheap biological waste material, viz. eggshells, for the development of hydroxyapatite (HAp) resins and evaluated them for protein purification. Initially, the calcite of the eggshell carbonate was converted into metastable vaterite microspheres. The HAp microspheres (ECHAp) were then prepared from eggshell carbonate microspheres using a novel dissolution-precipitation process. Synthetic source calcium carbonate was also used to prepare HAp microspheres (CHAp) for comparison. The purity and morphology of the apatite microspheres were characterized using X-ray diffraction (XRD) method, Fourier Transform Infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and laser diffraction particle analysis. Although both the apatites have similar morphology, the ECHAp has a larger surface area of 33.8 m2 g−1 compared to CHAp of 17.27 m2 g−1 by surface area analysis method. A commercial HAp matrix (CHT) with similar properties was also studied for comparison. All the apatite microspheres were found to have a similar protein binding capacity for bovine serum albumin (BSA). But ECHAp showed better protein separation for BSA and lysozyme mixture compared to CHAp and CHT matrices. The ECHAp matrix was also found to be highly stable over 20 purification cycles. Hence, the eggshell waste seems to have the potential for HAp matrix by a novel carbonate route with ease of preparation and also an economical packing material for chromatographic purification of biomolecules. © 2021 Elsevier Ltd and Techna Group S.r.l.