We have developed an open-source workflow that allows for quantitative single-cell analysis of organelle morphology, distribution, and inter-organelle contacts with an emphasis on the analysis of mitochondria and mitochondria-endoplasmic reticulum (mito-ER) contact sites. As the importance of inter-organelle contacts becomes more widely recognized, there is a concomitant increase in demand for tools to analyze subcellular architecture. Here, we describe a workflow we call MitER (pronounced "mightier"), which allows for automated calculation of organelle morphology, distribution, and inter-organelle contacts from 3D renderings by employing the animation software Blender. We then use MitER to quantify the variations in the mito-ER networks of Saccharomyces cerevisiae, revealing significantly more mito-ER contacts within respiring cells compared to fermenting cells. We then demonstrate how this workflow can be applied to mammalian systems and used to monitor mitochondrial dynamics and inter-organelle contact in time-lapse studies.