Preparation of UFM1‐Derived Probes through Highly Optimized Total Chemical Synthesis**

Ufmylation is involved in various cellular processes and associated with many human diseases. The understanding of this modification relies on the use of customized UFM1-derived probes for activity-based profiling of its related enzymes. This study presents a highly optimized total chemical synthesis for the generation of diverse UFM1-derived probes including UFM1-PA, Biotin-UFM1-PA and UFM1-AMC, in which a UFM1 C-terminal valine hydrazide was readily prepared by hydrazide-based ligation and used as a versatile handle for the installation of enzyme-sensitive warheads and fluorescent reporters. The resulting probes display high reactivity and selectivity for UFM1-specific enzymes in cell lysates. This strategy facilitates the generation and diversity of the UFM1-derived toolkit that can be employed to profile UFM1-specific enzymes, thereby shining insights into the dynamics of ufmylation.