As new approach methodologies (NAMs) are developed, there is a need for robust and efficient processes to establish scientific confidence in these approaches. To gain scientific confidence in NAMs, we can apply a framework that focuses on fitness for purpose, human biological relevance, and technical characterization. Using this framework, several critical differences in physiology, anatomy, and biochemistry between the rat and human respiratory tract become apparent and highlight the value of using human-relevant in vitro models to predict human responses. Because of these differences and the way they limit the ability of the rat to predict human effects, there has been a shift to develop and use human cell-based approaches to characterize potential portal-of-entry effects of inhaled substances. We describe the INSPiRE project, which assessed the value of in vitro models to predict the ability of chemicals to cause portal-of-entry effects on the human respiratory tract. To better understand the impact of experimental design, we assessed four chemicals from two distinct chemical classes in two different cell systems (BEAS-2B cells and MucilAirTM) using various exposure scenarios and biological endpoints. The results give important insights in how in vitro systems can be used to predict effects on the human respiratory tract and inform regulatory decision-making.