Chemical protein synthesis has emerged as a powerful approach for producing ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) in both their free and conjugated forms, particularly when recombinant or enzymatic strategies are challenging. By providing precise control over the assembly of Ub and Ubls, chemical synthesis enables the generation of complex constructs with site-specific modifications that facilitate detailed functional and structural studies. Ub and Ubls are central regulators of protein homeostasis, regulating a wide range of cellular processes such as cell cycle progression, transcription, DNA repair, and apoptosis. Ubls share an evolutionary link with Ub, resembling its structure and following a parallel conjugation pathway that results in a covalent isopeptide bond with their cellular substrates. Despite their structural similarities and sequence homology, Ub and Ubls exhibit distinct functional differences. Understanding Ubl biology is essential for unraveling how cells maintain their regulatory networks and how disruptions in these pathways contribute to various diseases. In this review, we highlight the chemical methodologies and strategies available for studying Ubls and advancing our comprehensive understanding of the Ubl system in health and disease.