Conventional biochemical analysis mainly focuses on the expression level of cellular proteins from entire cells. However, it has been increasingly acknowledged that the subcellular location of proteins often carries important information. Analysis of subcellular proteins conventionally requires subcellular fractionation which involves two steps: cell lysis to release proteins and high-speed centrifugation to separate the homogenate. Such approach requires bulky and expensive equipment and is not compatible with processing scarce cell samples of limited volume. In this study, we apply microfluidic flow-through electroporation to breach cell membranes and extract cytosolic proteins selectively in a single step. We demonstrate that this approach allows monitoring the translocation of the transcription factor NF-kappaB from the cytosol to the nucleus without the need of subcellular fractionation. Our technique is compatible with the processing of samples of various sizes and provides a simple and universal tool for bioanalytical analysis and spatial proteomics.