In this chapter, we present the materials and methods required to isolate and characterize circulating tumor cells (CTCs) from blood samples of cancer patients based on our newly developed microfluidic technologies. In particular, the devices presented herein are designed to be compatible with at\omic force microscopy (AFM) for post-capture nanomechanical investigation of CTCs. Microfluidics is well-established as a technology for isolating CTCs from the whole blood of cancer patients, and AFM is a gold standard for quantitative biophysical analysis of cells. However, CTCs are very scarce in nature, and those captured using standard closed-channel microfluidic chips are typically inaccessible for AFM procedures. As a result, their nanomechanical properties largely remain unexplored. Thus, given limitations associated with current microfluidic designs, significant efforts are put toward bringing innovative designs for real time characterization of CTCs. In light of this constant endeavor, the scope of this chapter is to compile our recent efforts on two microfluidic technologies, namely, the AFM-Chip and the HB-MFP, which proved to be efficient in isolating CTCs through antibody-antigen interactions, and their subsequent characterization using AFM.