We sought to elucidate the molecular roles of individual complex III subunits and assembly factors on complex III and respiratory chain supercomplex assembly, the effect on mitochondria and metabolism in general. For this a novel, comprehensive systems biology approach coupling gene-editing and mass-spectrometry has been applied. The CRISPR/Cas9 technology has been utilized to systematically target all nuclear encoded complex III subunits and known assembly factors. Individual gene-disrupted cell lines have been analysed and compared via BN-PAGE and metabolic profiling using the Seahorse Bioanalyzer. Stable isotope labelling with amino acids in cell culture (SILAC) and quantitative mass-spectrometry was performed to measure the relative levels of mitochondrial proteins across individual knockouts of subunits and assembly factors. We confirmed by parallel BN-PAGE analyses in combination with different detergents that the loss of individual complex III subunits leads to the complete degradation or destabilization of complex III into different sub-assembly classes. As a consequence, we observe destabilization of higher-order respiratory chain supercomplexes. Individual cell lines showed various levels of defects on mitochondrial function, and a shift towards glycolysis for energy production. Mass-spectrometry analyses of enriched mitochondria from individual subunit knockouts quantified the levels of ~70% of the known mitochondrial proteome in all cell lines, allowing us to understand the impact of the different classes of complex III dysfunction on complex assembly as well as the response made by various other mitochondrial biological processes. The presented data not only confirms but also expands on the pivotal role of complex III within the electron-transport chain, respiratory supercomplex and mitochondrial biogenesis. Insights into how the mitochondrial proteome responds to complex III dysfunction will potentially allow identification of novel complex III assembly factors which represent potential mitochondrial disease markers.