Mitochondrial oxidative phosphorylation (OXPHOS) disorders represent the most common genetic form of inherited metabolic diseases, affecting ~1/5000 births with a diagnosis rate of only ~60%. In addition, only very limited proven treatments are available and those fortunate enough to receive a molecular diagnosis often wait years. Our lab is developing the use of computational proteomics and metabolomics tools to complement existing diagnostic strategies, discover new disease genes, and understand the impact of mitochondrial dysfunction on a systems-wide level. The presented work will provide an update on our project to generate CRISPR/Cas9 gene disrupted cell lines of every known OXPHOS subunit and assembly factor – in total this represents >120 nuclear encoded genes. In particular I will be discussing the molecular roles of individual complex II and III subunits within wider mitochondrial function, as well as new data describing the roles of existing and novel assembly factors in respiratory chain complex assembly. Using quantitative mass-spectrometry analyses of mitochondria from individual knockout cell lines we quantified the levels of ~80% of the known mitochondrial proteome, allowing us to understand the impact of the different classes of complex II and III dysfunction on complex assembly, as well as the response made by various other mitochondrial biological processes. Knockouts could be grouped by hierarchical clustering, likely into structural ‘modules’ giving insights into assembly of both complexes. Complementary to this, I will present our latest metabolic profiling data demonstrating the unusual roles of complex II subunits and assembly factors on the tricarboxylic acid cycle (TCA cycle). The presented data not only confirms but also expands on the pivotal role of complex II/III within the electron-transport chain, respiratory supercomplex and mitochondrial biogenesis. Deeper interrogation of our data into how the mitochondrial proteome and metabolome responds to respiratory chain dysfunction will potentially allow the identification of novel assembly factors which represent potential mitochondrial disease markers.