In vitro test methods can be used to evaluate the effects of inhaled substances on the human respiratory tract. Scientists are increasingly using these tools due to interest in maximizing reliability and human-relevance, and therefore, the ability to protect human health. Among these in vitro models, reconstructed human respiratory epithelium (RHRE) is designed to mimic aspects of the biology of the human respiratory tract and to reflect mechanisms perturbed by different insults, including chemical exposure. In this paper, the human biological relevance of RHRE, in particular the MucilAir model, is assessed to gauge confidence in the use of RHRE-based test methods to evaluate respiratory effects. While the paper focuses on MucilAir, many of the key concepts also apply to other RHRE models. Key features of MucilAir, such as the presence of relevant primary human cells that produce mucus and have beating cilia, demonstrate its relevance to human biology. The model is compatible with a range of assays (such as those assessing cytotoxicity, cell viability, and cellular barrier integrity) that measure key events that may occur in humans following exposure. In this paper, we also present case studies of how these models have been used to predict the toxicity of inhaled substances. Overall, RHRE can be used to provide a quantitative, mechanism-based understanding of the potential effects of chemicals on the human respiratory tract.