Mitochondrial complex I (CI) is the largest multi-subunit oxidative phosphorylation (OXPHOS) protein complex. Recent availability of a high-resolution human CI structure, and from two non-human mammals, enabled predicting the impact of mutations on interactions involving each of the 44 CI subunits. However, experimentally assessing the impact of the predicted interactions requires an easy and high-throughput method. Here, we created such a platform by cloning all 37 nuclear DNA (nDNA) and 7 mitochondrial DNA (mtDNA)-encoded human CI subunits into yeast expression vectors to serve as both 'prey' and 'bait' in the split murine dihydrofolate reductase (mDHFR) protein complementation assay (PCA). We first demonstrated the capacity of this approach and then used it to examine reported pathological OXPHOS CI mutations that occur at subunit interaction interfaces. Our results indicate that a pathological frame-shift mutation in the MT-ND2 gene, causing the replacement of 126 C-terminal residues by a stretch of only 30 amino acids, resulted in loss of specificity in ND2-based interactions involving these residues. Hence, the split mDHFR PCA is a powerful assay for assessing the impact of disease-causing mutations on pairwise protein-protein interactions in the context of a large protein complex, thus offering a possible mechanistic explanation for the underlying pathogenicity.