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Here, we developed a novel ELISA-based assay for quantifying double-stranded intact siRNAs for in vivo pharmacokinetic analysis. The assay makes use of dual-labeled unmethylated or methylated siRNA, 5′-end-labeled on one strand with biotin (capture marker), and with dinitrophenol (detection marker), on the other end. This ELISA-based assay was linear over the range of 10–100 fmol/ml, with a sensitivity (5.4 fmol/ml) 629-fold higher than fluorometric quantification methods. The coefficient of variation (CV) of the ELISA quantification was 9.4% for intra-assay and 12.1% for inter-assay. The assay was specific for double-stranded siRNAs. The intensity of the detected signal was reduced to background levels in the presence of single-stranded RNA. The ELISA-based assay revealed that the levels of methylated forms of siRNAs after transfection into A549 and HeLa cells were significantly higher than those of unmethylated siRNA forms. Applying this assay to a study of the pharmacokinetic profiles of intravenously administered siRNAs, we found that the higher blood concentrations were achieved using the methylated form of siRNAs than unmethylated form. Moreover, methylated siRNAs complexed to DOTAP-based cationic liposomes showed significantly higher and prolonged blood concentration–time profile, with 2.2-fold lower clearance rate (0.11 ± 0.02 ml/min) as compared to the uncomplexed form. These results demonstrate the utility of an ELISA-based assay for evaluating chemically modified siRNAs and cationic delivery systems, particularly from a pharmacokinetic perspective.Simple and sensitive ELISA-based quantification assay was developed for pharmacokinetics of siRNAs administered in lipoplexes. This assay will be useful for evaluation of siRNA delivery systems from a pharmacokinetic perspective.