AbstractBackgroundTissue clearing is currently revolutionizing neuroanatomy by enabling organ-level imaging with cellular resolution. However, currently available tools for data analysis require a significant time investment for training and adaptation to each laboratory’s use case, which limits productivity. Here, we present FriendlyClearMap, an integrated toolset that makes ClearMap1 and ClearMap2’s CellMap pipeline easier to use, extends its functions, and provides Docker Images from which it can be run with minimal time investment. We also provide detailed tutorials for each step of the pipeline.FindingsFor more precise alignment, we add a landmark-based atlas registration to ClearMap’s functions as well as include young mouse reference atlases for developmental studies. We provide an alternative cell segmentation method besides ClearMap’s threshold-based approach: Ilastik’s Pixel Classification, importing segmentations from commercial image analysis packages and even manual annotations. Finally, we integrate BrainRender, a recently released visualization tool for advanced 3-dimensional visualization of the annotated cells.ConclusionsAs a proof of principle, we use FriendlyClearMap to quantify the distribution of the 3 main GABAergic interneuron subclasses (parvalbumin+[PV+], somatostatin+, and vasoactive intestinal peptide+) in the mouse forebrain and midbrain. For PV+neurons, we provide an additional dataset with adolescent vs. adult PV+neuron density, showcasing the use for developmental studies. When combined with the analysis pipeline outlined above, our toolkit improves on the state-of-the-art packages by extending their function and making them easier to deploy at scale.