In this study, the dielectrophoretic response of prostate tumor initiating cells (TICs) was investigated in a microfluidic system utilizing contactless dielectrophoresis (cDEP). The dielectrophoretic response of prostate TICs was observed to be distinctively different than that for non-TICs, enabling them to be sorted using cDEP. Culturing the sorted TICs generated spheroids, indicating that they were indeed initiating cells. This study presents the first marker-free TIC separation from non-TICs utilizing their electrical fingerprints through dielectrophoresis.