Silver nanoparticles (AgNPs) are highly promising components for the development of innovative biomedical products. However, a critical issue remains the insufficient deep and quantitative understanding of their fundamental physicochemical properties. These properties essentially govern the bioactivity of silver nanoparticles and, consequently, the success of their biomedical applications. Current characterization methods do not fully capture the complex nature of AgNPs, leaving key questions unresolved, such as detailed surface properties, dynamic interactions in real biological environments, long-term changes, and the release of silver ions—all factors that influence the toxicity and performance of the nanoparticles. This gap in knowledge complicates the reproducibility of experiments, comparison of results, and proper evaluation of potential health risks associated with their use. While advanced techniques such as Atomic Force Microscopy (AFM), Inductively Coupled Plasma (ICP) spectroscopy, and X-ray Photoelectron Spectroscopy (XPS) further significantly our understanding, they still do not fully meet all the demands for understanding silver nanoparticles. Specifically, these methods face limitations in monitoring the dynamic and complex interactions of nanoparticles within real biological settings, especially physicochemical properties that are linked to toxicity and also the biological. Therefore, despite their invaluable role, these techniques represent only part of the solution for the thorough understanding and assessment of the biomedical performance of AgNPs, highlighting the need for continued research to ensure their safe and efficient biomedical utilization.