The majority of mitochondrial proteins are nuclear-encoded, translated as cytosolic precursors and require import across the mitochondrial membranes for proper organelle function. Mitochondrial precursor proteins utilise different pathways and mitochondrial translocation machineries depending on their intra-organellar localisation. These pathways and machineries are well characterised in Saccharomyces cerevisiae, but poorly understood in humans. In yeast, the small TIM proteins function as chaperone proteins in the mitochondrial intermembrane space to facilitate the transfer of hydrophobic membrane proteins between translocation machines of the outer and inner membranes. However, the function of the human small TIM proteins and their contribution to human mitochondrial biology remain unclear. Furthermore, the yeast small TIM, Tim8, has two human homologues of unknown function: hTim8a and hTim8b. Our study focused on this family of proteins, with particular emphasis on characterising hTim8b. To interrogate hTim8b function in mitochondria, we analysed changes in the mitochondrial proteome of a CRISPR/Cas9-generated hTim8bKO cell line using quantitative mass spectrometry. hTim8bKO mitochondrial proteomics showed depletion of intermembrane space proteins, Complex IV assembly factors and proteins involved in sulfur metabolism. We hypothesise hTim8b maintains the oxidative environment required for protein folding in the intermembrane space by regulating metalloproteins involved in Complex IV assembly.