The human amniotic membrane (hAM) or amnion contains two principal types of cells: amniotic epithelial cells (hAECs) and amniotic mesenchymal stromal cells (hAMSCs), located in two distinct regions: the epithelium and the stromal layer. Emerging evidence suggests that both of them retain multipotent/pluripotent characteristics, making the amniotic membrane a promising and very attractive source of cells for regenerative medicine. Therefore, the isolation of hAECs and hAMSCs has recently received great interest; they can be released by differential enzymatic digestion and various procedures have been reported; however, significant contamination of hAMCs with hAECs and vice versa frequently occurs. This unit describes an efficient and rapid method to separate, mechanically, amniotic mesoderm from amniotic epithelium in order to obtain, after subsequent enzymatic digestions, purified population of hAMCs and hAECs. In this way, the cells can be cultured or investigated for other aims avoiding additional procedures related to their purification.