Introduction: OTULIN, OTUB1 and OTUB2 are deubiquitinases, the enzymes responsible for reversing ubiquitination process that occupies key roles in numerous cellular processes. The ubiquitination protein-protein interaction (PPI) network has been extensively explored in order to unravel the complexity of ubiquitin pathway. However, many significant challenges remain to develop a network-based understanding of the ubiquitination complexity including incompleteness of human interactome. Therefore, we aim to construct a pair of yeast two-hybrid (Y2H) vectors using pDEST32/pDEST22 vector system as a preparation for screening OTULIN-, OTUB1- and OTUB2-interacting proteins from human cDNA library, with ultimate aim of expanding the PPI network in human ubiquitome. Methods: OTULIN, OTUB1 and OTUB2 were cloned into entry vector using pCR™8/GW/TOPO® TA Cloning® system and shuttled into pDEST™32 bait vector by LR recombination reaction. To generate Y2H prey library clones, cDNA library was synthesized from HEK293 cells and cloned into donor vector pDONR™222 before transferred into destination vector pDEST™22. Results: DNA sequencing analysis confirmed the correct sequence of OTULIN, OTUB1 and OTUB2 inserts in pDEST32. Meanwhile, generation of cDNA library in pDEST22 produced 5.2 x 106 clones. Randomly picked pDEST22-cDNA clones showed that the recombination rate was 83% and gel electrophoresis indicated that the inserts length ranged from 0.45 to 3.4 kb. Conclusion: OTULIN, OTUB1, OTUB2 and cDNA library were successfully cloned into Y2H bait and prey vectors. The clones have been transfected into competent yeast Saccharomyces cerevisiae strain MaV203 and Y2H experiment to screen novel OTULIN-, OTUB1- and OTUB2-interacting protein from human cDNA library is underway.