Planarians are free-living flatworms that emerged as a crucial model system to understand regeneration and stem cell biology. The ability to purify neoblasts, the adult stem cell population of planaria, through fluorescence-activated cell sorting (FACS) has tremendously increased our understanding of pluripotency, specialization, and heterogeneity. To date, the FACS-based purification methods for neoblasts relied on nuclear dyes that discriminate proliferating cells (>2N), as neoblasts are the only dividing somatic cells. However, this method does not distinguish the functional states within the neoblast population. Our work has shown that among the neoblasts, the pluripotent stem cells (PSCs) are associated with low mitochondrial content and this property could be leveraged for purification of the PSC-enriched population. Using the mitochondrial dye MitoTracker Green (MTG) and the nuclear dye SiR-DNA, we have described a method for isolation of PSCs that are viable and compatible with downstream experiments, such as transplantation and cell culture. In this protocol, we provide a detailed description for sample preparation and FACS gating for neoblast isolation in planaria.