High-content screening (HCS) has gained interest in cellular imaging because of its ability to provide statistically significant data from multiple parameters simultaneously in cell-based assays. Although HCS has been mainly used in drug discovery, it has other potentially useful applications, such as elucidating the processes involved in nonviral gene vector-mediated gene delivery, as was explored in this study. HCS was used to measure transfection efficiency and cytotoxicities of polyplexes made from fluorescently labeled polyethylenimine (PEI) and pDNA encoding EGFP (pEGFP-N1). The results generated using HCS were confirmed using more conventional and labor-intensive methods. For the first time, a relationship between transfected cells and the number of polyplexes in the cytoplasm was shown. Four to five polyplex signals were found in the cytoplasm of successfully transfected cells, whilst nontransfected cells harbored less than one polyplex signal within the cytoplasm. HCS has the potential to be used as a tool in the field of gene delivery. HCS can not only simultaneously measure transfection efficiency and cytotoxicity of various nonviral gene vectors; it can also be used to track such vectors through various subcellular compartments.