Introduction: OTULIN, OTUB1 and OTUB2 are deubiquitinases, the enzymes responsible for reversing ubiquitina- tion 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-inter- acting 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 electro- phoresis 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 com- petent yeast Saccharomyces cerevisiae strain MaV203 and Y2H experiment to screen novel OTULIN-, OTUB1- and OTUB2-interacting protein from human cDNA library is underway.