This work reports the direct separation of twenty one Fmoc, Boc, Trt, and Pmc, single and double protected amino acids (PAA) using protein-based and macrocyclic antibiotic-based chiral columns. This group of PAAs represents a cross-section of commonly employed reagents in peptide synthesis with diverse structures and protecting groups. The Ultron Ovomucoid (OVM) chiral column was found to be extremely versatile in the resolution of these molecules without the need of any chemical modification. Compounds not separated in the Ovomucoid column, were resolved using Human serum albumin (HSA) and Chirobiotic T (Teicoplanin) columns.