Cancer, a metastatic disorder disruptsnormal growth and metabolic activities leads to Malignancy. Without signs and symptoms,prostate cancer is one increasing in men day by day and more than 1.1 million cases were recorded by 2012.It has been shown that basal epithelial cells are more potent in the conversion of PCaandsome of the luminal cells are castration resistant completely lost due to apoptotic cell death mechanism. After Androgen replacement therapy castration becomes a weapon for the identification of the altered mechanistic aversion of prostate cancer from stem cells. These cancer stem cells are the elusive therapeutic targets for present oncotherapies which are responsible for disease recurrence and metastases. The main aim of the present study is to isolate and characterize Prostate cancer stem cells based on prostate specific cell surface markers in prostate cancer tissues. In this study we have taken 6 prostate tissues and isolated CD 133 and CD 44 positive cells through Florescence staining method. Further isolation has done through MACS which has been cultured and taken from DMEM media. In conclusion confirmation of isolated cells as prostate cancer stem cells was done through western blot based on the identification of other markers OCT-4 and MDR-1.