Ubiquitin-like 3 (UBL3) acts as a post-translational modification (PTM) factor and regulates protein sorting into small extracellular vesicles (sEVs). sEVs have been reported as vectors for the pathology propagation of neurodegenerative diseases, such as α-synucleinopathies. Alpha-synuclein (α-syn) has been widely studied for its involvement in α-synucleinopathies. However, it is still unknown whether UBL3 interacts with α-syn, and is influenced by drugs or compounds. In this study, we investigated the interaction between UBL3 and α-syn, and any ensuing possible functional and pathological implications. We found that UBL3 can interact with α-syn by theGaussia princepsbased split luciferase complementation assay in cells and immunoprecipitation, while cysteine residues at its C-terminal, which are considered important as PTM factors for UBL3, were not essential for the interaction. The interaction was upregulated by 1-methyl-4-phenylpyridinium exposure. In drug screen results, the interaction was significantly downregulated by the treatment of osimertinib. These results suggest that UBL3 interacts with α-syn in cells and is significantly downregulated by epidermal growth factor receptor (EGFR) pathway inhibitor osimertinib. Therefore, the UBL3 pathway may be a new therapeutic target for α-synucleinopathies in the future.