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Microscopy is a fundamental technology driving new biological discoveries. Today microscopy allows a large number of images to be acquired using, for example, High Throughput Screening (HTS) and 4D imaging. It is essential to be able to interrogate these images and extract quantitative information in an automated fashion. In the context of neurobiology, it is important to automatically quantify the morphology of neurons in terms of neurite number, length, branching and complexity, etc. One major issue in quantification of neuronal morphology is the “crossover” problem where neurites cross and it is difficult to assign which neurite belongs to which cell body. In the present study, we provide a solution to the “crossover” problem, the software package NeuronCyto II. NeuronCyto II is an interactive and user‐friendly software package for automatic neurite quantification. It has a well‐designed graphical user interface …