SUMMARY: Segmentation of neural somata is a crucial and usually the most time-consuming step in the analysis of optical functional imaging of neuronal microcircuits. In recent years, multiple auto-segmentation tools have been developed to improve the speed and consistency of the segmentation process, mostly, using deep learning approaches. Current segmentation tools, while advanced, still encounter challenges in producing accurate segmentation results, especially in datasets with a low signal-to-noise ratio. This has led to a reliance on manual segmentation techniques. However, manual methods, while customized to specific laboratory protocols, can introduce variability due to individual differences in interpretation, potentially affecting dataset consistency across studies. In response to this challenge, we present ViNe-Seg: a deep-learning-based semi-automatic segmentation tool that offers (i) detection of visible neurons, irrespective of their activity status; (ii) the ability to perform segmentation during an ongoing experiment; (iii) a user-friendly graphical interface that facilitates expert supervision, ensuring precise identification of Regions of Interest; (iv) an array of segmentation models with the option of training custom models and sharing them with the community; and (v) seamless integration of subsequent analysis steps. AVAILABILITY AND IMPLEMENTATION: ViNe-Seg code and documentation are publicly available at https://github.com/NiRuff/ViNe-Seg and can be installed from https://pypi.org/project/ViNeSeg/.