Cyclic tetrapeptides comprise a potent and selective class of molecules with a wide range of biological activities, including the phytotoxic activity of tentoxin and the histone deacetylase (HDAC) inhibitory effects of chlamydocin. The incorporation of a functional aziridine group within cyclic peptides enables their conformational control and allows for late-stage and site-selective functionalization of these molecules, thereby creating the potential for covalent protein labeling. This protocol describes the solid-phase synthesis, cyclization, and site-specific structural modification of aziridine-containing tetrapeptides. The linear precursors are assembled by solid-phase peptide synthesis using Fmoc-protected amino acid building blocks, followed by head-to-tail peptide cyclization. Cyclization is performed using a slow reverse-addition method that prevents the formation of undesired higher-order cyclo-oligomeric side products. Site-specific structural modification of the resulting macrocycles is described using sodium azide or thiophenol as representative examples. It requires ∼4 d to prepare peptide macrocycles from their respective Fmoc-protected amino acid starting materials, an improvement upon the 3 weeks required for conventional solution-phase methods. This protocol also addresses important considerations regarding the handling of these compounds, whose electrophilic aziridine functionalities can otherwise be prone to undesired side reactions. With recent developments in aziridine-containing macrocyclic peptide synthesis and the potential for covalent protein labeling, these scaffolds represent a valuable addition to many screening libraries, and we expect that access to these macrocycles will facilitate efforts in drug discovery and molecular probe development.