A key to understanding biological response due to cell exposure to chemical constituents in aerosols is to accurately be able to determine the delivered dose. Deposition efficiency and uniformity of deposition was measured experimentally in the Vitrocell ® 24/48 air–liquid-interface (ALI) in vitro exposure system using monodisperse solid fluorescent particles with mass median aerodynamic diameters (MMAD) of 0.51, 1.1, 2.2 and 3.3 µm. Experimental results were compared with computational fluid dynamics (CFD; using both Lagrangian and Eulerian approaches) predicted deposition efficiency and uniformity for a single row (N = 6) of cell culture inserts in the Vitrocell ® 24/48 system. Deposited fluorescent monodisperse particles were quantified using fluorescent microscopy and Image J software. Experiments were conducted using a suspension of two particle MMADs with each experiment being conducted a total of three times on different days. The average experimentally measured deposition efficiency ranged from a low of 0.013% for 0.51 µm MMAD particles to a maximum 0.86% for 3.3 µm MMAD particles. There was good agreement between the average experimentally measured and the CFD predicted particle deposition efficiency (regardless of approach) with agreement being slightly better at the smaller MMADs. Experimentally measured and CFD predicted average uniformity of deposition was >45% of the mean and within 15% of the mean for 0.51 µm and 2.2 MMAD µm particles, respectively. Experimentally measured average uniformity of deposition was between 15 and 45% of the mean while CFD predictions were within 15% of the mean for 1.1 and 3.3 µm MMAD particles. The deposition efficiency and uniformity across the cell culture inserts for solid particles should be considered when designing exposure regimens using the Vitrocell ® 24/48 ALI in vitro exposure system.