Circulating tumor cells (CTCs), the “seeds” of fatal metastasis, intravasate into the bloodstream throughout the early stages of cancer, promoting the generation of micrometastatic reservoirs, some of which ultimately evolve to metastatic tumors. Though CTC enumeration has been invaluable in prognosticating metastatic cancer progression and response to treatment, therapeutic targeting of CTCs remains a future objective because signaling mechanisms of CTCs are largely unknown. In this chapter, we describe methods of CTC isolation, their cell surface antigenic and mutational signature validation, and CTCs’ comprehensive transcriptomic characterization. First, we highlight these concepts and unique CTC gene expression signatures by transcriptomic analyses, showing that CTCs harbor a significant proportion of mitotically inactive cells. Conversely, CTCs associated with breast cancer brain metastasis (BCBM) possess increased Notch signaling and immune-evasion pathways. Second, we generated an in vivo model of clinical metastatic dormancy by the systemic injection of CTC-enriched cell populations in immunodeficient mice (NSG), which revealed that mTOR signaling maintains dormancy in bone marrow-resident breast cancer cells (BMRTCs). Third, we developed a model of liver metastasis using sequential grafting of CTCs derived from triple-negative breast cancer patients and identified integrin signaling as a major pathway in liver metastasis. Lastly, by employing similar strategies in melanoma patient-derived CTCs and transcriptional profiling, we have reported that selective targeting of the USP7 pathway reduces micrometastatic burden in preclinical models. Collectively, these studies demonstrate the feasibility and reproducibility of undertaking CTC transcriptomic analyses. We foresee that the advent of single-cell sequencing/feature barcoding technologies will usher a new area and foster important discoveries in CTC transcriptomics to address unmet clinical needs.