On-chip cell sorting is a promising technique for sorting stem cells in culture. On-chip cell sorting allows minimization of lab personnel involvement in cells processing, dramatically reducing the risk of cell culture contamination. We developed a fluorescence-activated On-Chip Cell Culture Sorting (O3CS) system, which combines a biocompatible semiconductor light addressable microarray (chip) and optical setup for chip addressing and cell culture observation. The optical setup has fluorescent and reflected-light microscope capability for visualization and control of cell populations. High-resolution detection of 'unwanted' cells with a high-efficient sorter, based on light-induced electroporation is in the core of the O3CS implemented in NeuroSyntek StemOptimizer 6+. We demonstrated capability of the system to perform cell culture fluorescence activated sorting by inducing irreversible single-cell electroporation, validated O3CS sorting efficacy with fluorescent microscopy and flow cytometry, and compared it with the magnetic-activated cell sorting, demonstrating vastly superior performance in selectivity, efficiency, and sorting speed.