Nanocomposite hydrogels that contain silver nanoparticles (AgNPs) are especially attractive for various biomedical applications (e.g., antimicrobial wound dressings, coatings and soft tissue implants) due to strong antimicrobial activity of released silver nanoparticles and/or ions over prolonged times. However, all potential biomedical products have to be thoroughly specified fulfilling strict safety requirements. Characterization of nanocomposites is additionally complicated due to potential harmful effects of nanoparticles and accumulation in cells and tissues. This paper summarizes methods for preclinical characterization of hydrogel nanocomposites containing AgNPs with the particular attention on Ag/alginate hydrogels. Standard physicochemical characterization methods include transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM), UV-visible spectroscopy, and Fourier transform infrared spectroscopy (FTIR). Functional in vitro characterization relies on different methods for estimation of silver release, antimicrobial activity, and nanocomposite cytotoxicity. Here, we specially focus on utilization of 3D bioreactor systems that mimic native physiological environments with the aim to reliably predict nanocomposite behavior during implementation and so to decrease the need for animal experimentation. These systems were shown to provide more accurate and relevant data on silver release and cytotoxicity as compared to static systems such as 2D cell monolayer cultures. Finally, nanocomposites are evaluated in vivo in different animal models, which are in the case of wound dressings typically mice, rats, and pigs. The present review provides a basis for defining a strategy for comprehensive and efficient preclinical characterization of novel nanocomposites attractive not only for those containing AgNPs but also other metallic nanoparticles aimed for biomedical applications. Key points center dot A platform for devising comprehensive preclinical evaluation of nanocomposites. center dot Biomimetic bioreactors provide reliable functional nanocomposite evaluation. center dot Cells in 2D cultures are more sensitive to silver nanoparticles than in 3D cultures. center dot Biomimetic bioreactor 3D cell/tissue cultures can address the in vitro-in vivo gap.