Summary Both strands of human mitochondrial DNA (mtDNA) are transcribed in continuous, multi-genic units that are cleaved into the mature rRNAs, tRNAs, and mRNAs required for respiratory chain biogenesis. We sought to systematically identify nuclear-encoded proteins that contribute to processing of mitochondrial RNAs (mt-RNAs) within the organelle. First, we devised and validated a multiplex “MitoString” assay that quantitates 27 mature and precursor mtDNA transcripts. Second, we applied MitoString profiling to evaluate the impact of silencing each of 107 mitochondrial-localized, predicted RNA-binding proteins. With the resulting dataset, we rediscover the roles of recently identified RNA processing enzymes, detect unanticipated roles of known disease genes in RNA processing, and identify new regulatory factors. We demonstrate that one such factor, FASTKD4, modulates half-lives of a subset of mt-mRNAs and associates with mitochondrial RNAs in vivo. MitoString profiling may be useful in diagnosing and deciphering the pathogenesis of mtDNA disorders.