Human brain organoids are 3D self-assembling constructs obtained from human pluripotent stem cells, differentiating, and recapitulating the structural and functional complexity of the human brain. Quantitatively describing the cellular spatial arrangement within these constructs and their morphology is important to assess their structural resemblance to the human brain, and thus effectively demonstrating their ability to recapitulate the brain salient microstructural features. Thanks to pluripotent stem cell technology, brain organoids obtained from cells of healthy and diseased patients will also allow the study of different neuropathies, ranging from developmental to neurodegenerative disorders, at the individual level.In this light, here we adapt a pipeline, developed by our team, for acquiring and segmenting midbrain organoids, considered a promising model for studying Parkinson’s Disease at the microscale. Firstly, midbrain organoids are immunolabelled to reveal the Tyrosine Hydroxylase-positive dopaminergic neurons, whose degeneration leads to the disease progression. Then, the z-stacks acquired using a confocal microscope are segmented using SENPAI, an algorithm purposely developed to accurately isolate neurons thanks to a 4D clustering based on second derivatives and pixels intensity. Once cells are reconstructed in their native arrangement within the organoid, they are characterized by means of morphological parameters (Sholl analysis, Strahler analysis, shape detection) describing their shape, size, and complexity.Future development will include exploiting organoid technology and the pipeline developed to quantitatively identify any microstructural differences in dopaminergic neurons.