Direct exposure to inhaled mainstream cigarette smoke is known to cause smoking-related damage in the human lung. Aerosol exposure of human three dimensional (3D) organotypic airway epithelial tissue cultures, growing at the air-liquid interface is a well-established in vitro model enabling systems biology-based Reduced Risk Product (RRP*) assessment. The characterization of RRPs exposure is an essential part of the whole product assessment strategy. Smoke compounds such as nicotine or primary metabolites of smoke compounds (e.g. acrolein glutathione adducts) are assessed as exposure markers, which may be utilized for an insert-to-insert normalization of 3D tissue cultures. Moreover, the magnitude by which reduced glutathione (GSH) becomes depleted may also provide a valuable cellular stress marker when assessing the effect of RRP aerosol, compared to conventional cigarette smoke. Furthermore, investigations on the migration kinetics including adsorption and excretion occurring in 3D organotypic tissue cultures have been started to better understand the bioavailability of smoke compounds over time and to be able to link it to the observed impact on the exposed in vitro model. To better understand the role of xenobiotic metabolism after cigarette smoking, human subcellular liver and lung fraction models (microsomes, S9) have been established within a case study focused on phase I and phase II metabolism of furan type compounds to assess metabolite profiles and reactive metabolites relevant for toxicology. Finally, approaches using both subcellular fractions metabolism models and organotypic tissue cultures are in line with the 3R strategy to find an alternative for animal testing. The integration of the exposure characterization results together with the systems toxicology approach to measure the biological impact of the exposure has the potential to further highlight the mode of action associated to RRPs exposure. *Reduced-Risk Products ("RRPs") is the term we use to refer to products with the potential to reduce individual risk and population harm in comparison to smoking combustible cigarettes.