Whether silver nanoparticles (AgNPs) degrade and release silver ions (Ag(+)) in vivo has remained an unresolved issue. To evaluate the biodistribution and dissolution behavior of intravenously administered AgNPs in living rats, we employed a knotted reactor (KR) device to construct a differentiation scheme for quantitative assessment of residual AgNPs and their released Ag(+) ions in complicated animal tissues; to do so, we adjusted the operating parameters of the KR, namely, the presence/absence of a rinse solution and the sample acidity. After optimization, our proposed differentiation system was confirmed to be tolerant to rat tissue and organ matrix and provide superior reliability of differentiating AgNPs/Ag(+) than the conventional centrifugal filtration method. We then applied this differentiation strategy to investigate the biodistribution and dissolution of AgNPs in rats 1, 3, and 5 days postadministration, and it was found that the administered AgNPs accumulated predominantly in the liver and spleen, then dissolved and released Ag(+) ions that were gradually excreted, resulting in almost all of the Ag(+) ions becoming deposited in the kidney, lung, and brain. Histopathological data also indicated that toxic responses were specifically located in the AgNP-rich liver, not in the Ag(+)-dominated tissues and organs. Thus, the full-scale chemical fate of AgNPs in vivo should be integrated into future assessments of the environmental health effects and utilization of AgNP-containing products.