While the OECD test guidelines and mostly animal assays have been used to study the toxic effects of chemicals for many years, very little is known about the potential toxicity of vast majority of inhaled chemicals. Considering large number of chemicals and complex mixtures present in indoor and outdoor air, heavy reliance on animal test methods appear to be not adequate. Continuing scientific developments are needed to improve the process of safety evaluation for the vast number of chemicals and inhaled materials. The aim of this study was to optimise in vitro methods for toxicity testing of airborne contaminants. An integrated approach was designed in which appropriate exposure techniques were developed. A diversified range of in vitro assays using multiple human cell systems were implemented. Direct exposure of cells to airborne contaminants was developed by culturing cells on porous membranes in conjunction with a horizontal diffusion chamber system. Dose-response curves were generated allowing the measurement of toxicity endpoints. Toxicity ranking of test chemicals, based on obtained IC50 (50% inhibitory concentration) values, in different human cells and in vitro assays were determined. Airborne IC50 values were calculated for selected volatile organic compounds (xylene; 5350 ± 328 ppm > toluene; 10500 ± 527 ppm) and gaseous contaminants (NO2; 11 ± 3.54 ppm > SO2; 48 ± 2.83 ppm > and NH3; 199 ± 1.41 ppm). Results of this research indicate the significant potential of in vitro methods as an advanced technology for toxicity assessment of airborne contaminants.