Gold nanoparticles have attracted significant scientific and technological interest due to their ease of synthesis, chemical stability, and unique optical properties. They have potential biomedical applications in chemical sensing, biological imaging, drug delivery, and cancer treatment. However, their potential toxicity and health impact must be understood before clinical use. The interactions of gold nanoparticles with physiological fluids are crucial for understanding their biological effects and mitigating toxic effects. This Perspective discusses recent findings on the toxicity of gold nanoparticles in vitro and in vivo, and provides experimental recommendations for future research at the interface of nanotechnology and biological systems. Gold nanoparticles, especially nanorods, have unique optical properties due to localized surface plasmon resonance (LSPR), which allows them to absorb and scatter light in the visible and near-infrared range. These properties make them useful for biomedical applications such as photothermal therapy and imaging. The plasmon band position can be tuned by controlling the size and shape of the nanoparticles, enabling them to absorb in the biological "water window" of 800–1200 nm, where few chromophores absorb and water does not absorb, allowing deeper penetration into biological tissues. Gold nanoparticles can interact with biological media, leading to changes in their physiochemical properties such as size, aggregation state, surface charge, and surface chemistry. Protein adsorption to the nanoparticle surface can mediate cellular uptake via receptor-mediated endocytosis, influencing toxicity and uptake. The presence of proteins can change the effective surface charge of nanoparticles, affecting their interaction with cells. In vitro studies have shown that gold nanoparticles can be toxic, depending on their size, surface chemistry, and the cell type. For example, smaller gold nanoparticles (less than 2 nm) show different chemical reactivity compared to larger ones. The toxicity of gold nanoparticles can be influenced by the presence of surfactants such as CTAB, which can be toxic to cells at submicromolar doses. However, the toxicity of the supernatant (which contains no nanoparticles) was found to be similar to the whole nanoparticle solution, indicating that the toxicity may arise from the surfactants rather than the nanoparticles themselves. In vivo studies have shown that the toxicity of gold nanoparticles can vary depending on their size. Smaller nanoparticles (3–5 nm) and larger nanoparticles (50–100 nm) were not toxic at the doses used, while intermediate sizes (8–37 nm) caused severe sickness, weight loss, and shorter lifespan in mice. The toxicity of intermediate-sized nanoparticles was linked to major organ damage in the liver, spleen, and lungs. The discrepancy between in vitro and in vivo results highlights the importance of considering the complex biological environment when assessing nanoparticle toxicity. The biodistribution of gold nanoparticles in organisms is size-dependent. Smaller nanoparticles can cross biological barriers and enter cells, while larger nanoparticles are retained in organs such as the liver and spleen. The elimination of nanoparticles from the body is influencedGold nanoparticles have attracted significant scientific and technological interest due to their ease of synthesis, chemical stability, and unique optical properties. They have potential biomedical applications in chemical sensing, biological imaging, drug delivery, and cancer treatment. However, their potential toxicity and health impact must be understood before clinical use. The interactions of gold nanoparticles with physiological fluids are crucial for understanding their biological effects and mitigating toxic effects. This Perspective discusses recent findings on the toxicity of gold nanoparticles in vitro and in vivo, and provides experimental recommendations for future research at the interface of nanotechnology and biological systems. Gold nanoparticles, especially nanorods, have unique optical properties due to localized surface plasmon resonance (LSPR), which allows them to absorb and scatter light in the visible and near-infrared range. These properties make them useful for biomedical applications such as photothermal therapy and imaging. The plasmon band position can be tuned by controlling the size and shape of the nanoparticles, enabling them to absorb in the biological "water window" of 800–1200 nm, where few chromophores absorb and water does not absorb, allowing deeper penetration into biological tissues. Gold nanoparticles can interact with biological media, leading to changes in their physiochemical properties such as size, aggregation state, surface charge, and surface chemistry. Protein adsorption to the nanoparticle surface can mediate cellular uptake via receptor-mediated endocytosis, influencing toxicity and uptake. The presence of proteins can change the effective surface charge of nanoparticles, affecting their interaction with cells. In vitro studies have shown that gold nanoparticles can be toxic, depending on their size, surface chemistry, and the cell type. For example, smaller gold nanoparticles (less than 2 nm) show different chemical reactivity compared to larger ones. The toxicity of gold nanoparticles can be influenced by the presence of surfactants such as CTAB, which can be toxic to cells at submicromolar doses. However, the toxicity of the supernatant (which contains no nanoparticles) was found to be similar to the whole nanoparticle solution, indicating that the toxicity may arise from the surfactants rather than the nanoparticles themselves. In vivo studies have shown that the toxicity of gold nanoparticles can vary depending on their size. Smaller nanoparticles (3–5 nm) and larger nanoparticles (50–100 nm) were not toxic at the doses used, while intermediate sizes (8–37 nm) caused severe sickness, weight loss, and shorter lifespan in mice. The toxicity of intermediate-sized nanoparticles was linked to major organ damage in the liver, spleen, and lungs. The discrepancy between in vitro and in vivo results highlights the importance of considering the complex biological environment when assessing nanoparticle toxicity. The biodistribution of gold nanoparticles in organisms is size-dependent. Smaller nanoparticles can cross biological barriers and enter cells, while larger nanoparticles are retained in organs such as the liver and spleen. The elimination of nanoparticles from the body is influenced