Computational analysis of bio-images by deep learning (DL) algorithms has made exceptional progress in recent years and has become much more accessible to non-specialists with the development of ready-to-use tools. Study of oogenesis mechanisms and female reproductive success has also recently benefited from the development of efficient protocols for three-dimensional (3D) imaging of ovaries. Such datasets have a great potential for generating new quantitative data but are, however, complex to analyze due to the lack of efficient workflows for 3D image analysis. Here, we integrated two existing open-source DL tools, Noise2Void and Cellpose, into an analysis pipeline dedicated to 3D follicular content analysis and available on Fiji. Our pipeline was developed on larvae and adult medaka ovaries but was also successfully applied to different types of ovaries (trout, zebrafish and mouse). Image enhancement, Cellpose segmentation and label post-processing enabled automatic and accurate quantification of these 3D images exhibiting irregular fluorescent staining, low autofluorescence signal or heterogeneous follicles sizes. In the future, this pipeline will be useful for extensive cellular phenotyping in fish or mammals for developmental or toxicology studies.