Abstract: The method of one-step sequence- and ligation-independent cloning (SLIC) combined with FPLC-LC-MS/MS was adopted to find out the substrate protein which modified by Haloarcula hispanica’s ubiquitin-like protein ThiS in vivo. A N-terminal His₆ tag has been added to the ThiS with DMSO respiration to format multiple ubiquitylation conjugations. Based on SDS-PAGE analysis, the expression level of ubiquitylaion from plasmid is much higher than genome. From site mutation, the amino acid 89 of ThiS has been mutated from Serine to Lysine/Arginine. The purified ubiquitylation conjugation protein has been split up by trypsin and the modification sites were identified by LC-MS/MS analysis of GlyGly signatures(114 Da) on lysine side-chains of tryptic peptides derived from ThiS. The results show that the conjugates substrates of ThiS include 4 target proteins: MoaE, MsrA, MsrB and Fe-S cluster assembly protein SufB. Follow-up affinity purification of selected protein targets (Fe-S and MoaE) confirmed the LC-MS/MS results. This method combining the amino acid site mutation and MS/MS analytical approach is efficient and accurate for the detection of Ub/Ubl modification substrate protein, including the substrate conjugation site. This research can provide basic knowledge for the future study of ubiquitin/ubiquitin-like protein function in eukaryote cell.