Circulating tumor cells (CTCs) are malignant cells shed into the bloodstream from a tumor that have the potential to establish metastases in different anatomical sites. The separation and subsequent characterization of these cells is emerging as an important tool for both biomarker discovery and the elucidation of mechanisms of metastasis. Established methods for separating CTCs rely on biochemical markers of epithelial cells that are known to be unreliable because of epithelial-to-mesenchymal transition, which reduces expression for epithelial markers. Emerging label-free separation methods based on the biophysical and biomechanical properties of CTCs have the potential to address this key shortcoming and present greater flexibility in the subsequent characterization of these cells. In this review we first present what is known about the biophysical and biomechanical properties of CTCs from historical studies and recent research. We then review biophysical label-free technologies that have been developed for CTC separation, including techniques based on filtration, hydrodynamic chromatography, and dielectrophoresis. Finally, we evaluate these separation methods and discuss requirements for subsequent characterization of CTCs.