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To achieve efficient and safe cationic carrier-mediated gene delivery for gene therapy, the optimal ratio of carrier to DNA in formulations is a key factor and it is usually determined prior to transfection experiments. In this work, a simplified drop-and-read assay was developed for the first time using paper as a platform to estimate the equivalence ratio of cationic carriers to negatively charged DNA. By spotting a series of complexes containing varied ratios of carrier to DNA on filter paper, then allowing them to dry and finally dropping yellowish-green anionic 2′,7′-dichlorofluorescein dye solution on top of the complexes, the equivalence point was detectable by the instant formation of stable pink spots as a result of the dye adsorption onto the positively charged complexes and free carriers. The method gave the same results as those determined by gel retardation assay and zeta potential measurement, however it allowed more rapid reporting of results in 5 min and required no tedious steps, harmful reagents or expensive instruments. By using paper instead of microcentrifuge tubes and omitting centrifugation, plasticware and electrical energy were no longer consumed and disposal of this degradable material was more environmentally friendly. With respect to analytical performance, filter paper inherently holding negative charge helped to trap and concentrate the complexes on the white background, enabling greater visibility of the colored spots and a lower required amount of DNA used for the assay. The method was successfully applied to estimate the equivalence ratios in a variety of gene delivery formulations containing different types of cationic carriers, i.e. polymers, dendrimers, liposomes and niosomes.