With the growing use of nanomaterials in bioapplications, the nanotoxicity of new nanomaterials has become a safety concern when used in various applications. In this chapter, technical developments on carbon nanotubes are described including a historical account, experimental models and potential bioapplications.Carbon nanotube (CNT) materials display superior properties in electric current carrying capacity, thermal conductivity, and thermal stability. Due to the unique CNT structure with high-aspect ratio, CNT may show unusual toxicity and complicate its safe use in a target tissue. To test nanotoxicity of CNT, we describe a set of protocols of prior knowledgebased physical and chemical characteristics to develop 3-dimensional in vitro models of the intact skin, and a 3D in vitro model of the human airway using a co-culture of normal human bronchial epithelial cells and normal human fibroblasts. The human airway 3D model served as a tool of health risk assessment of CNTs on the human respiratory systems. To test functionality at different CNT concentrations in a 3D model, physical characteristics of multiwalled CNTs and production of nitric oxide (NO) served as cell viability and inflammatory marker; mitochondrial activity (MTT assay) served as the cytotoxic response of the epithelial cell layers; transepithelial electrical resistance (TER) measured nanotoxicity in the changes in airway physiological function. Cytoxicity and inflammatory responses of CNTs were dependent on different size, mass, shape, and functionality of CNTs as viable in vivo tests were conducted to evaluate the Ashutosh Tiwari and Atul Tiwari (eds.) Nanomaterials in Drug Delivery, Imaging, and Tissue Engineering,