Breast cancer is a leading cause of cancer-associated death in women. The clinical management of breast cancers is normally carried out using a combination of chemotherapy, surgery and radiation therapy. The majority of research investigating breast cancer therapy until now has mainly utilized two-dimensional (2D) in vitro cultures or murine models of disease. However, there has been significant uptake of three-dimensional (3D) in vitro models by cancer researchers over the past decade, highlighting a complimentary model for studies of radiotherapy, especially in conjunction with chemotherapy. In this review, we underline the effects of radiation therapy on normal and malignant breast cells and tissues, and explore the emerging opportunities that pre-clinical 3D models offer in improving our understanding of this treatment modality.