Airborne polycyclic aromatic hydrocarbons (PAHs) and their derivatives are of particular concern for population health due to their abundance and toxicity via inhalation. Lung toxicity testing includes exposing lung epithelial cell lines to PAHs in a culture medium containing inorganic species, lipids, proteins, and other biochemicals where the cell response is influenced among others by the toxic chemical accessibility in the medium. While inhalation bioaccessibility of PAHs and other toxicants was previously studied in surrogate lung fluids, studies measuring bioaccessibility in cell culture media are rare. In this work, a method was developed to characterize PAH bioaccessibility in a culture medium used for mouse lung epithelial (FE1) cells. Further, the optimised method was tested using commercially available standard reference material of urban particulate matter (PM) as well as polyurethane foam passive air samplers (PUF-PAS). The method provided a high precision and recovery of analytes, indicating no losses during sample processing and analysis. PAHs had non-linear concentration-responses, with the culture medium approaching saturation with PM concentration of 500 μg mL−1. The results showed that phenanthrene, a 3-ring PAH, was significantly more bioaccessible than ≥4-ring congeners in the culture medium (up to ∼2.5 folds; p < 0.05). Finally, using pre-deployed PUF-PAS from a residential and an industrial site, five PAHs were found in the culture medium, including naphthalene, phenanthrene, anthracene, fluoranthene, and pyrene. This work provides a proof of concept to enable future studies to assess the inhalation bioaccessibility of polycyclic aromatic compounds and other airborne pollutants collected using PUF-PAS.