Background In an era of precision medicine, there is a requirement for in vitro models that reflect the genetic, cellular and pathophysiological hallmarks of individual patients. Organoids, three-dimensional cultures that have been generated from primary tissue and remain genetically stable, provide a novel platform to investigate disease, although still lack important contributions from the microenvironment. Assembloids are recently developed co-cultures of patient–derived endometrial organoid that also include patient derived stromal cells arranged into three dimensional representations of the endometrium. In this study, we established endometrial assembloids to provide pre-clinical testing platforms for the investigation of endometrial biology, its relationship to endometrial pathophysiology and association with reproduction. Using assembloids, we found success in long-term culture and the recapitulation of endometrium in vitro. Methods Endometrial biopsies were collected from patients and epithelial and stromal cell isolated using standard size exclusion techniques. Epithelial cells were established as endometrial organoids and stromal cells cultured in standard 2-dimensional cell culture plates. Both epithelial and stromal cultures in various combinations were processed to individual cells and recombined inside Matrigel to establish three dimensional assembloids. Assembloids were grown for 10 days and identity and microenvironment influence characterised by whole mount immunofluorescence. Results Recombination of endometrial organoids and stromal cells established a 3D structure of epithelial glands surrounded by stromal cells recapitulating the endometrial environment for over 10 days in culture. Immunofluorescence confirmed the expression of progenitor cells indicating models remained capable of differentiation into various cell states. Both estrogen and progesterone receptor were present in both epithelial and stromal cells, confirming the in vitro model remained hormonally responsive. Conclusions In conclusion, we demonstrated that the production of endometrial assembloids that closely recapitulates the characteristics of parental tissue and can be used for in vitro models of endometrial and reproductive disorders.