SummaryThe lack of a suitable explant culture system restricts the study of factors secreted by the mouse placenta into maternal circulation. Here, we present a protocol for culturing the endocrine junctional zone of the mouse placenta, free from the decidua and labyrinthine layers in serum-free medium. We describe steps for dissecting and separating layers, dicing tissue, and culture setup. We then detail medium processing for downstream analysis. This model allows the investigation of placental signals that may regulate maternal physiology.For complete details on the use and execution of this protocol, please refer to Yung et al. (2023).1