Due to the substantial role of cancer stem cells (CSCs) in therapeutic resistance and metastasis, the study of them is of great importance. However, their low abundance within the tumor cells makes it challenging to study their molecular pathophysiology. This limitation highlighted the critical need for reliable enrichment techniques to provide enough cells for in vitro tests. This review focuses on various methods that implemented for enrichment of cancer stem-like cells’ subpopulations from the heterogeneous population of cancer cells. In this regard, various key physicochemical parameters such as hypoxia, shear stress, high glucose concentration, autophagy, and chemotherapy-induced stress have been explored for their potential to enhance CSC enrichment. The results presented in the literature confirmed that modifying these parameters can improve the enrichment of CSCs by promoting stem-like cells phenotype. However, there is still a necessity for comprehensive studies that compare the impact of these parameters on CSC enrichment, which should be addressed in future works.