SUMMARY: MyelinJ is a free user friendly ImageJ macro for high throughput analysis of fluorescent micrographs such as 2D-myelinating cultures and statistical analysis using R. MyelinJ can analyse single images or complex experiments with multiple conditions, where the ggpubr package in R is automatically used for statistical analysis and the production of publication quality graphs. The main outputs are percentage (%) neurite density and % myelination. % neurite density is calculated using the normalize local contrast algorithm, followed by thresholding, to adjust for differences in intensity. For % myelination the myelin sheaths are selected using the Frangi vesselness algorithm, in conjunction with a grey scale morphology filter and the removal of cell bodies using a high intensity mask. MyelinJ uses a simple graphical user interface and user name system for reproducibility and sharing that will be useful to the wider scientific community that study 2D-myelination in vitro. AVAILABILITY AND IMPLEMENTATION: MyelinJ is freely available at https://github.com/BarnettLab/MyelinJ. For statistical analysis the freely available R and the ggpubr package are also required. MyelinJ has a user guide (Supplementary Material) and has been tested on both Windows (Windows 10) and Mac (High Sierra) operating systems. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.