Assessing the safety of inhaled substances in the alveolar region of the lung requires an understanding of how the respired material interacts with both physical and immunological barriers. Human alveolar-like macrophages in vitro provide a platform to assess the immunological response in the airways and may better inform the understanding of a response to an inhaled challenge being adaptive or adverse. The aim of this study was to determine if a morphometric phenotyping approach could discriminate between different inhaled nicotine products and indicate the potential mechanism of toxicity of a substance. Cigarette smoke (CS) and e-liquids extracted into cell culture medium were applied to human alveolar-like macrophages in mono-culture (ImmuONE™) and co-culture (ImmuLUNG™) to test the hypothesis. Phenotype profiling of cell responses was highly reproducible and clearly distinguished the different responses to CS and e-liquids. Whilst the phenotypes of untreated macrophages were similar regardless of culture condition, macrophages cultured in the presence of epithelial cells were more sensitive to CS-induced changes related to cell size and vacuolation processes. This technique demonstrated phenotypical observations typical for CS exposure and indicative of the established mechanisms of toxicity. The technique provides a rapid screening approach to determine detailed immunological responses in the airways which can be linked to potentially adverse pathways and support inhalation safety assessment.