The U.S. Food and Drug Administration has regulatory authority over tobacco products, including conventional cigarettes and next generation products (NGPs) such as e-cigarettes and tobacco heating products (THPs). There is a desire by the industry, regulators and animal protection organizations to incorporate non-animal test methods for tobacco product and NGP assessment. When assessing respiratory effects in vitro, reliable exposure systems that deliver aerosols to cellular/tissue cultures (such as human reconstructed airways or lung slices) at the air–liquid interface are needed. Using nicotine dosimetry, we report the characterization of a Vitrocell VC1 in our laboratories (IIVS, USA). Nicotine, generated from a 3R4F reference cigarette or NGP (e-cigarette and THP) aerosols at source and the exposure interface (culture media), was assessed using ultra-high-performance liquid chromatography–tandem mass spectrometry. These data were compared to published dosimetry data for the same products, generated at a different laboratory (BAT R&D, Southampton, UK), on different exposure systems (VC10 and Borgwaldt RM20S) to confirm repeatability.