We aimed to improve the efficiency of isolating endometrial epithelial and stromal cells (EMECs and EMSCs) from hysterectomy specimens. We revealed by immunohistochemical staining that the large tissue fragments remaining after collagenase treatment, which are usually discarded after the first filtration in the conventional protocol, consisted of glandular epithelial and stromal cells. Therefore, we established protease-treatment and cell suspension conditions to dissociate single cells from the tissue fragments, and isolated epithelial (EPCAM-positive) and stromal (CD13-positive) cells by fluorescence-activated cell sorting. Four independent experiments showed that, on average, 1.2 x 10^6 of EMECs and 2.8 x 10^6 EMSCs were isolated from one hysterectomy specimen. We confirmed that the isolated cells presented transcriptomic features highly similar to those of epithelial and stromal cells obtained by the conventional method. Our improved protocol facilitates future studies to better understand the epigenetic regulation underlying the dynamic changes of the endometrium during the menstrual cycle. Although protocols for the isolation of endometrial stromal and epithelial cells (EMSCs and EMECs) have been well established, the number of EMECs obtainable by the current protocols is relatively small. To improve the efficiency of isolating EMECs as well as EMSCs from endometrial tissues, we established protease-treatment and cell suspension conditions to efficiently dissociate single cells from endometrial tissue fragments, and isolated epithelial (EPCAM-positive) and stromal (CD13-positive) cells by fluorescence-activated cell sorting. By conducting a microarray-based transcriptome analysis, we confirmed that the cells isolated by the modified protocol developed in this study maintain the transcriptomic properties of endometrial epithelial and stromal cells.