Direct assessment of patient samples holds unprecedented potential in the treatment of cancer. Circulating tumor cells (CTCs) in liquid biopsies are a rapidly evolving source of primary cells in the clinic and are ideal candidates for functional assays to uncover real-time tumor information in real-time. However, a lack of routines allowing direct and active interrogation of CTCs directly from liquid biopsy samples represents a bottleneck for the translational use of liquid biopsies in clinical settings. To address this, we present a workflow for using a microfluidic vortex-assisted electroporation system designed for the functional assessment of CTCs purified from blood. Validation of this approach was assessed through drug response assays on wild-type (HCC827 wt) and gefitinib-resistant (HCC827 GR6) non-small cell lung cancer (NSCLC) cells. HCC827 cells trapped within microscale vortices were electroporated to sequentially deliver drug agents into the cytosol. Electroporation conditions facilitating multi-agent delivery were characterized for both cell lines using an automatic single-cell image fluorescence intensity algorithm. HCC827 GR6 cells spiked into the blood to emulate drug-resistant CTCs were able to be collected with high purity, demonstrating the ability of the device to minimize background cell impact for downstream sensitive cell assays. Using our proposed workflow, drug agent combinations to restore gefitinib sensitivity reflected the anticipated cytotoxic response. Taken together, these results represent a microfluidics multi-drug screening panel workflow that can enable functional interrogation of patient CTCs in situ, thereby accelerating the clinical standardization of liquid biopsies.