Chiral ordered mesoporous silica (COMS) was synthesized in the presence of amino acid proline by combining tetraethyl orthosilicate and quaternized aminosilane silica sources. The as-prepared materials were activated by calcination or microwave chemical extraction to remove the organic templates. The powder X-ray diffraction and N2 adsorption characterization revealed in COMS the structural and textural features of MCM-41-type silica. The chirality of the material was disclosed by mixed and separate l- and d-proline adsorption on the COMS prepared with l-proline (l-Pro-COMS) and d-proline (d-Pro-COMS). It was found that the maximum l-proline and d-proline adsorption capacities on l-Pro-COMS were ca. 2.3 and 0.6 mmol/g, respectively, while the adsorption of d-proline was higher than that of l-proline on d-Pro-COMS. Finally, both activation routes yielded enantioselective silicas able to separate proline racemate.