Proper protein regulation is crucial for cell function. Ubiquitin is a 76 amino acid (aa) protein functioning as a post translational modification and thereby playing a central role in protein regulation. Ubiquitin (Ub) is covalently conjugated to the target protein by ubiquitin E3 ligases in a process known as ubiquitination and removed by deubiquitinating enzymes (DUBs) in a process known as de-ubiquitination. The DUB ubiquitin C-terminal Hydrolase 1 (UCHL1) is one of the most abundant proteins in the brain, constituting 1–2% of the total soluble protein, and is highly expressed in neurons. Variants of UCHL1 are associated with neurodegenerative disease, but its role stays elusive. Thus, uncovering the role of UCHL1 in MNs will contribute to a better understanding of neurodegenerative disease and possibly initiate the discovery of novel therapies. In this project an initial mapping of candidate binding partners of UCHL1 in human motor neurons is presented with the final goal to get insight into the role of UCHL1 in brain context. To achieve this, human induced pluripotent stem cells (hiPSC) derived from healthy control, patients harboring point mutations in uchl1, and an hiPSC UCHL1 KO cell line generated via gene editing, were further differentiated into motor neurons (MNs). Prior to differentiation, the quality of hiPSC clones was evaluated using standard assays assessing gene expression and protein expression patterns of pluripotent markers, as well as their in vitro trilineage differentiation ability. Furthermore, successful differentiation of MNs was confirmed by evaluating expression of specific markers at genome and protein level at three maturation stages: motor neuron progenitor cells (MNP) after 14 days of culture, MNs after 21 days of culture, and MNs after 31 days of culture. MN at the most mature stage (MN after 31 days of culture) were submitted to chemical in vivo crosslinking with formaldehyde and used for isolation of UCHL1 protein complexes by co-immunoprecipitation (co-IP) followed by quantitative mass-spectrometry (MS) analysis. By subtracting background proteins (identified on the KO sample), 14 proteins were uniquely identified or enriched in the healthy control MNs, representing potential UCHL1 binding partners in these cells. The functions of the proteins were diverse including chromatin remodeling, transcription regulation, translation, cell development, proliferation, cell survival, neural signaling, clearance, metabolism and mitochondrial function, DNA repair and immunity, and regulation of cytoskeleton. Using the same approach, 27 proteins were identified in patient MN sample. These candidate proteins did also show diverse functionality, including regulation of mitochondrial function via modulation of electron transport chain, modulation of mitochondrial translation, and mitochondrial DNA repair. Cellular tasks include chromatin remodeling, transcriptional regulation, translation, cell proliferation, development, and synaptic placidity, structure and transport, transmembrane transporter, E3 ubiquitin-protein ligase, purine nucleotide synthesis, and immunity. Proteins associated with UCHL1 in the healthy control sample highly differed from proteins associated with mutated UCHL1, suggesting that these mutations impact UCHL1 function both by promoting interactions to new partners and impairing binding to the usual ones. The only protein enriched in both healthy control and patient samples is the mitochondrial protein COX18, which promotes insertion of membrane proteins in the mitochondrial inner membrane, thereby being essential to maintain mitochondria integrity. Whether UCHL1 interacts directly to COX18 or is part of the same complexes containing COX18 both in control and patient MN and what are the functional consequences of such gain/loss interactions in mutated cells, remain to be revealed. The initial mapping of candidate proteins identified in this project lay the foundation for elucidation of identification of bona fide UCHL1 binding partners in future projects and provides initial data on the functional consequences of UCHL1 dysfunction at molecular level.