Updated publication reference for PubMed record(s): 32161263, 27626371. Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain (RC) and is composed of 44 different subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with RC complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species in the cell and its dysfunction is associated with mitochondrial disease, Parkinson’s disease and aging. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function, however the role and requirement of the remaining 30 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the energetic cost to the cell and has necessitated the involvement of a suite of assembly factors for complex I biogenesis. We used gene-editing to generate human knockout cell lines for each accessory subunit. We found that 25 subunits are strictly required for assembly of a functional complex and one subunit is essential for cell viability. Quantitative proteomic analysis of all 30 cell lines revealed that subunits belong to discrete complex I modules, and their loss affects the stability of subunits within these modules. Known assembly factors were found to correlate with most modules. Analysis of proteomic changes when other modules were destabilised revealed ATP5SL and DMAC1 as novel factors required to assemble the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with large-scale proteomics represents a powerful tool for dissecting large multi-subunit complexes and enabling the study of complex dysfunction at a cellular level.