Electroporation is widely used for drug and gene delivery with the aid of electric pulses. However, its mechanism is not fully discovered, and as a result, trial-and-error optimization process is required to identify proper electric conditions for every cell type system. By using optical tweezers technology, we are able to move and affixed single or multiple cells at desirable locations during electroporation. This provides the opportunity for in situ monitoring and quantifying the electroporation process at single or multiple cell level. We investigate the process for cell membrane polarization and pore formation as well as the uptake of therapeutic molecules and its intracellular transport. DNA dynamics and DNA-cell interactions are also quantified under different conditions, such as the relative positions between cells, cell to electrodes, between electrodes and the geometry and positions of electrodes. The possible influence of laser on the cell and cell membrane is isolated from electroporation and quantified. Our experimental observations are used as input for FEM models of cell electroporation and preliminary comparison is made. This study provides a new route to investigate the fundamental mechanism of nucleic acid transfer and intracellular response in electroporation. It is highly valuable to eliminate or simplify the optimization process of electroporation conditions for a new cell type, especially primary cells and stem cells.