Hydroxyapatite (HAp) chromatography is one of the classical chromatography methods in biomolecule separations and has been used to separate recombinant proteins, antibodies, viral particles, and DNAs. HAp is a calcium-phosphate complex Ca10(PO4)6(OH)2, composed of two sites, C-site due to Ca and P-site due to PO4. It is regarded as a mixed-mode or multi-modal chromatography since it involves unique electrostatic interaction (ion-exchange) and metal affinity. Several studies have shown that HAp chromatography enabled good separation performance of difficult separation, such as removing aggregates from monoclonal antibodies. Thus, it is a highly recommended golden standard for final polishing after Protein A chromatography. However, commercially obtained hydroxyapatites are of synthetic origin and high cost, so researchers earnestly look for alternative sources for producing hydroxyapatite resin to meet the demand. Hence, the utilization of biological wastes as a source of hydroxyapatite preparation is of great interest. In the present investigation, we utilized a cheap biological waste material, viz. eggshells, to develop hydroxyapatite (HAp) resins and evaluated them for various biomolecules purification.