AbstractWe report a PDMS microfluidic platform for parallel single‐cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or non‐invasive analysis schemes are performed. First, we report single‐cell trapping in a fast (2–5 min) and reproducible manner with a single‐cell capture yield of 85% using two cell lines (P3x63Ag8 and MCF‐7), employing a protocol which is scalable and easily amenable to automation. Following this, a mixed population of P3x63Ag8 and MCF‐7cells is stained in situ using the nucleic acid probe (Hoechst) and a phycoerythrin‐labeled monoclonal antibody directed at EpCAM present on the surface of the breast cancer cells MCF‐7 and absent on the myeloma cells P3x63Ag8 to illustrate the potential of the device to analyze cell population heterogeneity. Next, cells are porated in situ using chemicals in a reversible (digitonin) or irreversible way (lithium dodecyl sulfate). This is visualized by the transportation of fluorescent dyes through the membrane (propidium iodide and calcein). Finally, an electrical protocol is developed for combined cell permeabilization and electroosmotic flow (EOF)‐based extraction of the cell content. It is validated here using calcein‐loaded cells and visualized through the progressive recovery of calcein in the side channels, indicating successful retrieval of individual cell content.