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An open access and user friendly 3D automatic quantitative analysis tool for single plane illumination microscopy (SPIM) data. OpenSegSPIM is designed to extract quantitative relevant information from SPIM image stacks, such as the number of nuclei/cells and provide quantitative measurement (volume, sphericity, distance, intensity) on light sheet microscopy (LSM) images. Typically, it is a useful quantitative analysis tool for different biological samples such as neurospheres, zebrafish embryos, drosophila embryos, skin sample, mouse embryos and other organotypic cell culture. The OpenSegSPIM pipeline consists of a succession of step each one being enabled once the previous step has been completed. To provide a simple interface OpenSegSPIM minimizes the number of parameters without loss of segmentation quality. Furthermore, parameter input is an interactive process. For example, image smoothing is done in a few steps and the user only needs to measure the diameter of the nuclei. To facilitate the difficult step of parameter tuning the intermediate results are visualized in real time and can be evaluated.