Despite significant advances in analytical techniques, quantitative in situ characterization of silver nanoparticles (AgNPs) in biological systems—particularly regarding the dynamic balance between ionic and particulate silver—remains a major challenge. This study reveals that the dissolution behavior of AgNPs strongly depends on particle size and the surrounding biological medium. For example, 50?nm PVP-coated AgNPs exhibited significantly greater dissolution than 75?nm particles in both aqueous solution and DMEM medium. In aqueous environments, over 95% of silver from 50?nm NPs existed as ionic silver, compared to only about 38% from 75?nm NPs. In DMEM, a dynamic equilibrium was established, characterized by the concurrent dissolution of primary particles and the formation of new particulate species, leading to continuous fluctuations in particle number and ionic silver concentration over time. After 48?h of incubation, the released ionic silver accounted for approximately 38.4% from 50?nm particles and 26.2% from 75?nm particles. Chemical speciation analysis via synchrotron radiation-based X-ray absorption near edge structure (XANES)spectroscopy further demonstrated that intracellular silver underwent progressive transformation from the original AgNPs into Ag?S nanoparticles, reaching a conversion ratio of 61.9% at 12?h, along with minor formation of AgCl. This transformation was closely linked to the acidic intracellular milieu and interactions with biological ligands. Although no marked cytotoxicity was observed within the first 24?h of exposure, the gradual intracellular accumulation of transformation products, particularly Ag?S nanoparticles, eventually led tomild cytotoxic effects. These findings collectively underscore that the biological impact of AgNPs is fundamentally governed by their intracellular chemical transformation dynamics.