Endometriosis affects 5–10% of women in reproductive age and causes pelvic pain and subfertility. Exact etiology of the disease is unknown. Here, we present a microfluidic platform for characterizing mechanical properties of eutopic endometrial stromal cells of endometriosis patients based on cellular deformability inside narrow microchannels. Primary human endometrial stromal cells were isolated from eutopic endometrium of endometriosis patients (4407 cells, from 7 endometriosis patients) and from disease-free women (4541 cells, from 6 control women) and were pumped through microchannels (formed of polydimethylsiloxane (PDMS) by standard soft lithography, with dimensions of 8 × 20 × 150 μm, as width × height × length) at a constant flow rate of 2 μL/min. High-speed imaging was used to capture videos of cells as they flow inside microchannels, and a computer vision code was used to track cells, measure their area, and calculate the time each cell takes to pass through the microchannel. Compared with their counterparts from control women, eutopic endometrial stromal cells from endometriosis patients showed significantly increased deformation index (1.65 ± 0.2 versus 1.43 ± 0.19, respectively, P value < 0.001), and higher velocity in travelling through narrow microchannels (96.530 ± 0.710 mm/s versus 57.518 ± 0.585 mm/s, respectively, P value < 0.001). The same difference in velocities between the two cell types was maintained after controlling for cell area. Eutopic endometrial stromal cells of endometriosis patients showed a mechanical phenotype characterized by high deformability and reduced stiffness. This mechanical signature can represent basis of a mechanical biomarker of endometriosis.