Mitochondria are dynamic organelles playing essential metabolic and signaling functions in cells. Their ultrastructure has largely been investigated with electron microscopy (EM) techniques, which provided a wide range of information on how mitochondria acquire a tissue-specific shape, how they change during development, and how they are altered in disease conditions. However, quantifying protein-protein proximities using EM is extremely challenging. Superresolution microscopy techniques as direct stochastic optical reconstruction microscopy (dSTORM) now provide a fluorescent-based alternative to EM with a higher quantitative throughput. Recently, super-resolution microscopy approaches including dSTORM led to valuable advances in our knowledge of mitochondrial ultrastructure, and in linking it with new insights in organelle functions. Nevertheless, dSTORM is currently used to image integral mitochondrial proteins only, and there is little or no information on proteins transiently present at this compartment. The cancer-related Aurora kinase A/AURKA is a protein localized at various subcellular locations, including mitochondria. After performing dSTORM, we here use the Geo-coPositioning System (GcoPS) image analysis method to quantify the degree of colocalization of AURKA with compartment-specific mitochondrial markers. We show that two-color dSTORM provides sufficient spatial resolution to visualize AURKA in the mitochondrial matrix. We conclude by demonstrating that optimizing fixation procedures is a key step to follow AURKA in the matrix. In this light, we show that a methanol-based fixation leads to a better detection of the matrix pool of AURKA than an aldehyde-based fixation. Our results indicate that dSTORM coupled to GcoPS colocalization analysis is a suitable approach to explore the compartmentalization of non-integral mitochondrial proteins as AURKA, in a qualitative and quantitative manner. This method also opens up the possibility of analyzing the proximity between AURKA and its multiple mitochondrial partners with exquisite spatial resolution, thereby allowing novel insights into the mitochondrial functions controlled by AURKA.