Mineral oil aromatic hydrocarbons (MOAHs) include mutagenic and carcinogenic substances and are considered a potential health risk. Current methods address the total MOAH content but cannot address the actual toxicological hazard of individual components. This work presents a combined methodology closing those gaps: high-performance liquid chromatography (HPLC) coupled to gas chromatography with flame ionization detection was used to determine the MOAH content. To characterize present substance classes, comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry was applied. Preparative HPLC separated MOAHs into subgroups, which were tested with a miniaturized Ames test evaluating DNA reactivity of isolated fractions. Combining these methods allowed a correlation between present subgroups and DNA reactivity. The developed approach was applied to a mineral oil and distinguished between not DNA-reactive mono-and diaromatics and DNA-reactive triand polyaromatics, providing a proof of concept. Hereinafter, it will be applied to diverse sample matrices including mineral oils, food, and food contact materials.