Efficient metabolic engineering and the development of mitochondrial therapeutics often rely upon the specific and strong import of foreign proteins into mitochondria. Fusing a protein to a mitochondria-bound signal peptide is a common method to localize proteins to mitochondria, but this strategy is not universally effective with particular proteins empirically failing to localize. To help overcome this barrier, this work develops a generalizable and open-source framework to design proteins for mitochondrial import and quantify their specific localization. By using a Python-based pipeline to quantitatively assess the colocalization of different proteins previously used for precise genome editing in a high-throughput manner, we reveal signal peptide-protein combinations that localize well in mitochondria and, more broadly, general trends about the overall reliability of commonly used mitochondrial targeting signals.