Osmotic stress is detrimental for the plant which survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic mediated stress. Plants use the ubiquitin proteasome system to modulate protein contents required to respond to and tolerate adverse growth conditions and a role for ubiquitin to mediate endocytosis and trafficking of plant plasma membrane proteins has recently emerged. In this study, using the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry, a list of 786 ubiquitinated lysine (K-Ub) residues in 451 proteins of which 50 % were transmembrane proteins was compiled for Arabidopsis root membrane proteins, increasing the database of ubiquitinated substrates in plants. Ubiquitination particularly occurred within membrane proteins and the protein machinery involved in their internalization and sorting. Despite absence of identification of a strict ubiquitination motif, the presence of acidic residues close to K-Ub was evidenced. In silico interactomics analysis allowed to suggest two E2 ligases, UBC32 and UBC34 to target ubiquitination of membrane proteins. This study also showed a decrease of NRT2;1 with osmotic stress treatment, putatively mediated by its decreased ubiquitination, correlating with an increased NO3- influx. Then, this study revealed that a major plasma membrane aquaporin (PIP2;1) harbors two ubiquitination sites that may interfere with N-terminal acetylation and C-terminal phosphorylation, to putatively contribute to a decreased root hydraulic conductivity.