Abstract Study question Is OSIS Ovary (a three-dimensional ultrasound-based online system for automated ovarian follicle measurement) a reliable tool to track follicle growth during ovarian stimulation cycles? Summary answer We can conclude that OSIS Ovary constitutes a reliable follicle-measurement tool What is known already Ultrasound follicle tracking is a crucial part of cycle monitoring. When the number of follicles increases, the manual measurement process can become tedious, imprecise and can potentially compromise the clinical outcomes The use of automated volumetric software for follicle monitoring makes it possible to overcome these manual limitations, regardless of the moment in the cycle when the metrics are obtained and assessed, providing flexibility for both clinicians and patients At the same time, more objective metrics are obtained with automated volume segmentation than with manual follicle measurement Study design, size, duration A prospective cohort study was performed among 89 patients undergoing ovarian stimulation during in vitro fertilization or oocyte vitrification cycles They were invited to participate in the study between May 2020 and February 2021. All participants signed an informed consent form prior to the start of the study Participants/materials, setting, methods 534 ovarian ultrasound volumes and manual datasets were obtained from ovarian stimulation cycles from the 89 participant patients Three transvaginal (2D and 3D) ultrasounds scans were performed on volunteer patients on the three clinically relevant monitoring days: 4th- 5th day (first follicular assessment), 6th -7th day (introduction of the GnRH antagonist) and 8th-9th day (oocyte pickup scheduling) The mean follicle diameter and the follicle count were estimated manually (2D) and automated (3D with Osis Ovary) Main results and the role of chance Based on the mean follicle diameter and follicle count values obtained, the Pearson/intraclass correlation coefficients were 0.976/0.987 and 0.804/0.889 in ≥ 10mm follicles, 0.989/0.994 and 0.809/0.867 in ≥ 13mm follicles and 0.995/0.997 and 0.791/0.840 in ≥ 16mm follicles. The mean difference (MnD) for the mean diameter and follicle count was,respectively, 0.759/0.161 in ≥ 10mm follicles, 0.486/1.033 in ≥ 13mm follicles and 0.784/0.486 in ≥ 16mm follicles. The upper and lower limits of agreement (ULA and LLA) were 3.641/2.123 and5.392/3.070 in ≥ 10mm follicles, 3.496/2.522 and 4.285/2.218 in ≥ 13mm follicles, and3.723/2.153 and 2.432/1.459 in ≥ 16mm follicles. The limits of agreement range (LoAR) were5.764/8.462 in ≥ 10mm follicles, 6.048/6.503 in ≥ 13mm follicles and 5.876/3.891 in ≥ 16mm follicles Limitations, reasons for caution We can conclude that OSIS Ovary constitutes a reliable follicle-measurement tool. The performance observed can be considered very good and it must be progressively clinical studied to be assessed and optimized in the next future Wider implications of the findings Cloud-based features, such as multi-user functioning in any 3D ultrasound machine, could make OSIS Ovary an attractive resource for reproductive medicine clinics, making it easier to track follicle growth during ovarian stimulation cycles Trial registration number not applicable