In this study, we generated the UFSP1KO/UFSP2KO HEK293T cell line and validated it as the most suitable cellular model for the screening, identification, and confirmation of UFMylated substrate proteins. Furthermore, exogenous expression of the E3 ligase components UFL1 and DDRGK1 significantly enhances protein UFMylation levels, enabling the large-scale enrichment of UFMylated substrates.