Nanomaterials have received considerable recent attention because of their unique properties and diverse applications in technology and life sciences. However, the nano-structures of these materials possess very different physical and chemical properties compared to their larger size counterparts. Given the increasing use of nanoparticles in a range of products, it has become crucial to develop a fundamental understanding of the biological effects of nanoparticle exposure. Many in vitro studies have suggested that nanoparticles are cytotoxic and genotoxic; however, results are inconsistent, mainly due to interference between nanoparticles and dye molecules commonly used in traditional cytotoxicity assays. The purpose of this study was to set up a systematic method for evaluating biological effects of nanogold utilizing the xCELLigence System, an electrical impedance-based, dye-free, real-time system, to continuously monitor the dynamic effects of nanogold on cell growth, additionally, in the hope of establishing a biomarker signature that is relatively specific to gold nanoparticle exposure. The inhibitory effects of nanogold on the growth of numerous cell lines demonstrated using this system were also supported by several traditional in vitro toxicity assays, including MTS, trypan blue exclusion, colony forming assays. Mechanistic studies revealed that the action of nanogold is mediated by apoptosis induction or cell cycle delay, depending on cell type and cellular context and concentration of nanogold. Additional evidence is provided from our DNA microarray analysis suggesting that many proteins involved in DNA damage responses and cell cycle regulation are significantly up-regulated or down-regulated. In this study, we present a comprehensive overview of AuNP-induced cytotoxicity in a variety of mammalian cell lines, comparing several cytotoxicity assays. Collectively, these assays offer convincing evidence of the cytotoxicity of AuNPs and support the value of a systematic approach for analyzing the toxicology of nanoparticles. We performed a comprehensive gene expression profiles following exposure to AuNPs showed significant increase in the expression of approximately of 1500 genes (fold change >2; P <0.05), compared to the control group. The results presented in this study should provide helpful guidance on the future use of gold nanoparticles in occupational and medical applications.