Nonviral gene carriers must associate with and become internalized by cells in order to mediate efficient transfection. Methods to quantitatively measure and distinguish between cell association and internalization of delivery vectors are necessary to characterize the trafficking of vector formulations. Here, we demonstrate the utility of nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled oligonucleotides for discrimination between bound and internalized gene carriers associated with cells. Dithionite quenches the fluorescence of extracellular NBD-labeled material, but is unable to penetrate the cell membrane and quench internalized material. We have verified that dithionite-mediated quenching of extracellular materials occurs in both polymer- and lipid-based gene delivery systems incorporating NBD-labeled oligonucleotides. By exploiting this property, the efficiencies of cellular binding and internalization of lipid- and polymer-based vectors were studied and correlated to their transfection efficiencies. Additionally, spatiotemporal information regarding binding and internalization of NBD-labeled gene carriers can be obtained using conventional wide-field fluorescence microscopy, since dithionite-mediated quenching of extracellular materials reveals the intracellular distribution of gene carriers without the need for optical sectioning. Hence, incorporation of environmentally sensitive NBD-oligos into gene carriers allows for facile assessment of binding and internalization efficiencies of vectors in live cells.