Peptide backbone amide substitution can dramatically alter the conformational and physiochemical properties of native sequences. Although uncommon relative to N-alkyl substituents, peptides harboring main-chain N-hydroxy groups exhibit unique conformational preferences and biological activities. Here, we describe a versatile method to prepare N-hydroxy peptide on solid support and evaluate the impact of backbone N-hydroxylation on secondary structure stability. Based on previous work demonstrating the β-sheet-stabilizing effect of α-hydrazino acids, we carried out an analogous study with N-hydroxy-α-amino acids using a model β-hairpin fold. In contrast to N-methyl substituents, backbone N-hydroxy groups are accommodated in the β-strand region of the hairpin without energetic penalty. An enhancement in β-hairpin stability was observed for a di-N-hydroxylated variant. Our results facilitate access to this class of peptide derivatives and inform the use of backbone N-hydroxylation as a tool in the design of constrained peptidomimetics.