Although naphthenic acids are minor constituents in petroleum, their characterization is crucial because they are geochemically important tracers and play a key role in corrosion processes in refineries. Moreover, different isomers may exhibit different reactivity and may serve as potential biomarkers. However, determination of acid isomers in petroleum/hydrocarbon matrixes remains analytically challenging. Here, we achieve the separation and structural characterization of isomeric homologue series of naphthenic acids in petroleum samples by ion mobility time-of-flight mass spectrometry (IM-TOF MS).