Human Tim8a and Tim8b are mitochondrial proteins that constitute part of a chaperone network in the intermembrane space of mitochondria. It has been assumed that both hTim8 isoforms function in the import of nascent membrane proteins, by extrapolation of previous research performed in Baker’s yeast, Saccharomyces cerevisiae. The two human isoforms are the result of a metazoan-specific gene duplication and are differentially expressed across tissue-types. Furthermore, loss-of-function mutations in hTim8a result in a neurodegenerative condition called Mohr-Tranebjærg syndrome. In this study, we used HEK293 kidney cells and SH-SY5Y neuronal-like cells to model the tissue-types in which hTim8a and hTim8b are differentially expressed and created CRISPR/Cas9-mediated knock-out lines in both cell-types. A proteomics-led approach identified a loss of subunits and assembly factors for Complex IV upon loss of the hTim8 proteins in their relevant cell-types. Further biochemical analyses showed the hTim8 knock-out cell lines lacked complete assembly of mature Complex IV and a decreased cell viability. We identify Cox17, a copper chaperone for Complex IV, as a crucial player in hTim8-dependent assembly and hypothesise that loss of this interaction in our knock-out cell models blocks subassemblies of Complex IV from integrating into the mature Complex IV. We propose the loss of Complex IV function leading to increased neuronal cell death as the pathomechanism underlying Mohr-Tranebjærg syndrome.