BACKGROUND Small rodents continue to be the mainstay for the assessment of pharmacological and toxicological data of inhaled therapeutics. For meaningful interpretation of the results information about deposition of aerosol particles in the respiratory tract is warranted, but not trivial to obtain for animals with nose-only aerosol exposure. The purpose of this study was to develop and evaluate a general method to characterize the deposition of inhaled test particles in an in vitro model (IVR) of the rat's respiratory tract. METHODS A highly detailed, realistic and representative image using micro-CT scanning technology was obtained and the generated morphological data was used to construct a plastic replica of the average rat respiratory tract. The model was connected to a rodent ventilator, which allowed the breathing frequency (f, min(-1)) and tidal volume (V(T), mL) to be varied as required. Polydisperse fluorescent microsphere particles with an average mass median aerodynamic diameter (MMAD) of 3.1 μm and geometric standard deviation (GSD) of 2.2 μm were used as model compound. RESULTS Comparison of the experimental data for total and regional deposition levels with predicted outputs using the in silico MPPD model showed reasonably good relative agreement between the two models. The predictions were closest to the experimental values when default respiratory conditions of f=102 breaths/min and V(T) of 2.0 mL were used. Moreover, the IVR model revealed good correlation with published in vivo data. CONCLUSION Using the IVR model allows an easy, fast and reasonably precise estimation of the inhaled dose in rodent inhalation studies. The IVR has the potential to be used along with live rats in inhalation exposure studies, and thus provides the unique possibility to run an internal standard for dose deposition in the respiratory tract in each inhalation experiment. This should contribute to enable a greater understanding of drug pharmacokinetics and dynamics in rats and may improve dose extrapolation from animals to humans.