Ubiquitination is one of the most important post-translational modifications (PTMs) and involves the covalent attachment of ubiquitin to a lysine residue on a target protein. Despite ubiquitination playing a crucial role in regulating cellular processes, the ubiquitinated proteome has not been studied extensively in recombinant Chinese hamster ovary (CHO) cells. Moreover, ubiquitination modification in CHO cells is likely to have an impact on protein function related to the efficient productivity of biopharmaceuticals. In this chapter, we describe a comprehensive protocol for ubiquitin di-Glycine (diGly) peptide enrichment using an immunoprecipitation method from recombinant CHO cell proteins followed by Liquid chromatography-Mass spectrometry (LC-MS) analysis of the ubiquitinated proteome. The methods described are also applicable to differential ubiquitinated proteomic studies.