Mitochondria are crucial for cells, supplying up to 90% of the energy requirements for neurons. Their correct localisation is crucial and ensured by a transport system. Mitochondrial trafficking in neurons is particularly critical, because mitochondria must leave the soma and travel along the axon and dendritic network to facilitate neuronal function. Abnormal mitochondrial trafficking has been reported in several neurological disorders, therefore the ability to quantify and analyse mitochondrial trafficking is vital to improving our understanding of their pathogenesis. Commercial software currently lacks an automated approach for performing such quantitation. Here we demonstrate the development of the Mitochondrial Trafficking and Distribution (MiTrakD) analysis toolset, which consists of simple and free-to-use instructions for mitochondrial trafficking analysis using time-lapse microscopy. MiTrakD utilises existing Fiji (ImageJ) tools for semi-automated, fast and efficient analysis of mitochondrial trafficking and distribution, including velocity, abundance, localisation and distance travelled in neurons. We document MiTrakDs efficiency and accuracy by analysing mitochondrial trafficking using two-dimensional fluorescence images of cortical neurons of wild type mice after 6 days (DIV6), 10 days (DIV10) and 14 days (DIV14) of in vitro incubation. Using MiTrakD we have demonstrated that neurons at all developmental stages exhibited the same percentage of mobile mitochondria, all of which travel in equidistance. Interestingly, the mitochondria in neurons at DIV10 were in greater abundance and were faster than those at DIV6 and DIV14. We can also conclude that MiTrakD is more efficient than manual analysis and is an accurate and reliable tool for performing mitochondrial trafficking analysis in neuronal cells.