The study and investigation of cancer stem cells (CSCs) or tumour initiating cells (TICs) have received enormous attention over the 10 years. CSCs are rare, quiescent and capable of self-renewing and maintaining tumour growth and heterogeneity. Better understanding of CSCs will no doubt lead to a new era of both basic and clinical cancer research, reclassification of human tumours and development of novel therapeutic strategies. Therefore, the biological properties of CSCs, the relevance of CSCs to cancer therapy and methodologies to identify them are essential in order to address real and efficacious therapeutic strategies to eradicate the cancer. Here, we describe the main protocols to identify CSCs starting from primary tumours including glioblastoma, sarcoma, lung and breast cancers.