Mitochondria are the main source of energy in eukaryotic cells, oxidizing sugars and fats to generate ATP through oxidative phosphorylation, which is accomplished by the respiratory chain. Complex I deficiency is the most common mitochondrial disease and most often results in multi-system disorders with a fatal outcome. In humans, Complex I is composed of 44 different subunits where it forms a ~1MDa L-shaped structure in the mitochondrial inner membrane. Fourteen subunits are designated as “core” ones, based on their sequence conservation with bacterial subunits, and their catalytic role in Complex I function. While the roles of most of the other 30 “accessory subunits” are not clear, a number are thought to be involved in supporting the assembly of the complex. To investigate the importance of each of these accessory subunits in Complex I biogenesis, we generated individual human cell lines lacking expression of each accessory subunit through TALEN-mediated gene editing. We subsequently determined the effect of subunit loss on mitochondrial function and Complex I assembly. Furthermore we performed quantitative, shotgun proteomic analyses to investigate changes in the mitochondrial proteome and to generate profiles to assign related groups of accessory subunits. Through this, we were also able to identify novel proteins that respond to changes in mitochondrial dysfunction and commence their characterization.