Considerable　evidence　points　to　cancer　stem-like　cells　(CSCs)　as　responsible　for　promoting　progression,　metastasis,　and　drug　resistance.　Without　damage　to　the　cell　biological　properties,　single-cell-derived　tumor-sphere　is　encouraging　options　for　CSCs　identification　and　studies.　Although　several　single　cell-based　microfluidic　methods　have　been　developed　for　CSCs　studies,　clarifying　liaison　between　the　biomechanics　of　cells　(such　as　size　and　deformability)　and　stem　(such　as　tumor-sphere　formation　and　drug　resistance)　remains　challenging.　Herein,　we　present　a　platform　of　integrated　microfluidics　for　the　analysis　of　single-cell-derived　tumor-sphere　formation　and　drug　resistance.　Tumor-spheres　derived　from　different　biomechanics　(size　and/or　deformation)　single-cells　could　be　formed　efficiently　using　this　device.　To　demonstrate　the　microfluidic-platform　capability,　a　proof-of-concept　experiment　was　implemented　by　evaluating　single-cell-derived　sphere　formation　of　single　glioblastoma　cells　with　different　biomechanics.　Additionally,　a　course　of　chemotherapy　to　study　these　single-cell-derived　spheres　was　determined　by　coculture　with　vincristine.　The　results　indicate　that　tumor　cell　biomechanics　is　associated　with　single-cell-derived　spheres　formation;　that　is,　smaller　and/or　more　deformable　tumor　cells　are　more　stem-like　defined　by　the　formation　of　single-cell-derived　spheres　than　more　prominent　and/or　lesser　deformable　tumor　cells.　Also,　tumor-spheres　derived　from　single　small　and/or　more　deformable　tumor　cell　have　higher　drug　resistance　than　more　prominent　and/or　less　deformable　tumor　cells.　Our　device　offers　a　new　approach　for　single-cell-derived　sphere　formation　according　to　tumor　cell　different　biomechanical　properties.　Furthermore,　it　offers　a　new　method　for　CSC　identification　and　downstream　analysis　on　a　single-cell　level.　©　2019　American　Chemical　Society.