Few new inhaled medicines for the treatment of asthma and chronic obstructive pulmonary disease (COPD) have made it to market in the past decade despite the increasing global health burden of airways diseases. One of the reasons there has been limited success, despite considerable investment in this area to develop new inhaled drugs, is due to the observation of foamy macrophage responses in pre-clinical rat studies, which questions their safety for use in humans. The aim of this work was to develop in vitro cell culture assays to better characterise alveolar macrophage responses and to ascertain interspecies differences between human and rat macrophage models. Baseline profiles for cell health, morphology and lipid content parameters were generated for U937 (human) and NR8383 (rat) macrophage models using high content assays and analysis platform. Cells were then exposed to drug challenges with established pathological responses for assay validation. The rat and human in vitro macrophage models investigated had identical baseline profiles for all parameters tested. Typical response profiles were observed for both cell lines exposed to amiodarone (inducer of phospholipidosis) and staurosporine (apoptotic agent). These results indicate that the high content assays developed are suitable for understanding macrophage responses at the individual cell level, which may allow the characterisation of specific types of foamy macrophage responses. Future work is ongoing to further validate the assays to ascertain its potential, as an early pre-clinical screening tool, to predict the safety of candidate inhaled medicines.