The human ovary, pivotal to the endocrine and reproductive systems, undergoes dynamic structural changes throughout life, but the molecular mechanisms of puberty and menopause from a spatiotemporal aspect remain poorly characterized. This knowledge gap hinders advancements of infertility treatments, fertility preservation, and personalized medicine, which is of particular importance for childhood cancer survivors. Given that current options are limited to cryopreservation of ovarian tissue—a method developed for adult ovaries—a deeper understanding of ovarian cellular dynamics and cell type interactions across all ages is imperative. To date, there are no reports of live births following the transplantation of ovarian tissue collected during early infancy. The use of single-cell transcriptomics (scRNA-seq) has improved our understanding of ovarian biology and revealed exciting gene and protein candidates for genes and proteins involved in aging or treatment-induced damage. However, there is no clear understanding of the distribution of cells within the tissue, nor is there any distinction between follicle subpopulations. This study aims to construct a comprehensive spatiotemporal reference map of the human ovarian cortex, utilizing advanced transcriptomic and proteomic technologies, with a focus on unraveling the impact of chemotherapy and age-related changes. We analyzed a unique cohort of samples from pediatric patients undergoing fertility preservation (both treated and untreated), reproductive-age adults undergoing either gender reassignment surgery or cesarean section, and post-menopausal women. The goal was to understand patterns of cell localization, proportion changes, and associations with clinical features (age shift or treatment exposure). Utilizing a high-parameter fluorescent-based multiplex immunohistochemistry pipeline, we leveraged the expertise of the Human Protein Atlas (HPA) to identify five main cell types: oocytes, granulosa cells, immune cells, endothelial cells, and Schwann cells. This multiplexed immunofluorescence imaging provided a holistic view of the spatial organization of ovarian cortex across different age and therapy groups. Next, protein signatures for novel populations or subgroups of cells identified by scRNA-seq were stained alongside the multiplex panel outlining the five main cell types. Spatial analysis, based on the nearest neighbor method, unraveled the organization of the ovarian cortex, providing, for the first time, a spatiotemporal map of the human ovary across lifespan and treatment exposures. An in-depth view of this complex proteomic landscape offers a novel perspective that may shed light on uncharted territory and lead to breakthroughs in clinical interventions and therapeutics.