Silver nanoparticles have been widely used in the field of nanomedicine. A comprehensive understanding of their pharmacokinetics is crucial for proper risk assessment and safe biomedical applications. The purpose of this study was to investigate the safety of silver nanoparticles by determining their potential toxicity following 28 days of administration in Sprague-Dawley rats. The silver nanoparticles were administered by intravenous injection at the doses of 100, 200 and 500 μg/kg body weight for 28 consecutive days. Animals in the control group were received sterile water for injection. Each group consists of 10 male and 10 female rats. No treatment-related effects were seen in any of the parameters monitored in rats given 100, 200 and 500 μg/kg body weight/day of silver nanoparticles. The study proved that the use of up to 500 μg/kg body weight biosynthesized silver nanoparticles have no toxic effect on the target organs and found safe. However, the safety of the nanoparticles might be attributed to the covering of biological moieties on nanoparticles. Hence, the biofunctionalized nanoparticles can be safely used by selecting the required size and dose in medicines and drug delivery systems.