Current methodologies available to quantify changes in mitochondrial turnover are limited to pulse-chase assays or specific assays that quantify mitophagy. Accordingly, new tools that can assess mitochondrial turnover are needed for the study of cellular, subcellular, and spatial parameters of mitochondrial turnover and quality control. Recently, a group of studies described the use of the MitoTimer fluorescent probe to investigate various aspects of mitochondrial turnover, including changes to protein import, interorganelle protein sharing, and autophagy-mediated turnover. MitoTimer provides a fluorescent readout which directly relates to the mitochondrial turnover rate and allows quantification of relative changes to turnover. Importantly, MitoTimer can be used to investigate mitochondrial turnover on the subcellular level. Due to the fact that MitoTimer is a dual-emission probe and a number of factors can affect MitoTimer readout, certain considerations must be taken into account when using this tool both in experimental design and data interpretation. When used and interpreted appropriately, MitoTimer serves as a unique tool to understand pivotal aspects of mitochondrial turnover.