Previous work has shown that mitochondria play a critical role in priming stem cells to self-renew and proliferate. Here, we describe a protocol for enriching and identifying the mitochondria-primed stem cells (mpSCs) for their characterization and applications. We describe steps for enriching mpSCs with the environmental carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin in a skin keratinocyte lineage and for identifying mpSCs using single-cell transcriptomics and single-cell microscopy analyses of expression of relevant stem cell markers. For complete details on the use and execution of this protocol, please refer to Spurlock et al.1 Display omitted •Enriching “mitochondria-primed” neoplastic skin stem cells with TCDD exposure•Identifying neoplastic skin stem cells by single-cell transcriptomics•Identifying “mitochondria-primed” neoplastic skin stem cells by single-cell microscopy Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Previous work has shown that mitochondria play a critical role in priming stem cells to self-renew and proliferate. Here, we describe a protocol for enriching and identifying the mitochondria-primed stem cells (mpSCs) for their characterization and applications. We describe steps for enriching mpSCs with the environmental carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin in a skin keratinocyte lineage and for identifying mpSCs using single-cell transcriptomics and single-cell microscopy analyses of expression of relevant stem cell markers.