Accurate assessment of follicle number in mouse ovaries is essential for evaluating the impact of endogenous and exogenous insults on female fertility. The current gold standard for counting follicles is stereology, which is an expensive, time consuming, 2D, section-based histological technique that provides only limited information about the spatial organisation of follicles in the 3D ovary. To overcome these issues, our long-term goal is to develop a fast and accurate automated method to evaluate follicle number and 3D location in whole mouse ovaries. The aim of this pilot study was to determine if follicles could be fluorescently-labelled, imaged and counted in whole ovaries with normal follicle numbers, and following follicle depletion. Female post-natal day 20 C57/Bl6 mice received 2 weekly doses of cyclophosphamide (150mg/kg, n=1) to deplete follicles, or saline (n=2) to retain normal follicle numbers. Ovaries were fixed (4% paraformaldehyde) and optically cleared using the aqueous-immersion-based CUBIC method. Oocytes were labelled by incubating ovaries with anti-cKit and anti-Vasa antibodies, followed by AlexaFluor™-647 and -568 conjugated secondary antibodies, respectively, with DAPI for nuclear staining. Ovaries were imaged via the 10x objective on a Leica Stellaris5 standard confocal and a 3i Mariannas Spinning Disk confocal microscope, then Imaris software was used for image analysis and follicle counting. Follicles at all stages of development could be clearly easily visualised, throughout the entire depth of the ovary (~1.5mm), with fewer follicles present in the ovary exposed to cyclophosphamide compared to the saline treated ovaries. After wholemount analysis, cleared ovaries were re-fixed in Bouin’s, embedded in resin, and every 3rd 20µm section stained with PAS for stereology. Remarkably, tissue morphology was excellent and stereology is underway to determine if follicle numbers obtained from whole ovaries are comparable to the gold-standard. This study provides preliminary evidence indicating that follicle counting in whole ovaries is feasible.