To advance methods for bioaccumulation assessment of organic substances in air‐breathing organisms, the present study developed an in vitro approach for screening neutral hydrophobic organic substances for their bioaccumulation potential in air‐breathing organisms consisting of (1) depletion assays for chemicals in rat liver S9 subcellular fractions, (2) in vitro–in vivo extrapolation, and (3) whole‐organism bioaccumulation modeling to assess the biomagnification potential of neutral organic substances in the rat. Testing of the in vitro method on 14 test chemicals of potentially biomagnifying substances showed that the bioassays could be conducted with a high level of reproducibility and that in vitro–derived elimination rate constants were in good agreement with in vivo–determined elimination rate constants in the rat. Exploring the potential of the in vitro approach for screening organic chemicals for bioaccumulation in air‐breathing organisms indicated that chemical substances that exhibit a depletion rate constant in the S9 in vitro bioassay ≥0.3 h−1 are not expected to biomagnify in rats independent of their octanol–water partitioning coefficient (KOW) or octanol–air partitioning coefficient (KOA). The high level of reproducibility achieved in the test, combined with the good agreement between in vitro–derived and in vivo–determined depuration rates, suggests that the in vitro approach in combination with a KOA‐ and KOW‐based screening approach has good potential for screening chemicals in commerce for their bioaccumulation potential in air‐breathing organisms in a cost‐effective and expedient manner, especially if the bioassay can be automated. Environ Toxicol Chem 2022;41:2565–2579. © 2022 SETAC