Heavy oils are characterized by high proportions of asphaltenes, a class of polyaromatic hydrocarbons whose structures remain unknown. Previous work based on asphaltene physical-chemical properties and extensive spectroscopic and computational methods has generated controversy about two possible general conformations: island and archipelago. To address this controversy, this study employed a systematic integrative approach involving ion mobility (IMS), theoretical calculations, ultra-high resolution mass spectrometry (UHR MS), and pyrolysis gas chromatography coupled to high-resolution mass spectrometry (Py-GC-HR MS) to evaluate the composition and structure of asphaltenes. Two Brazilian oils with different API grades (heavy and light) were analyzed and compared. The comparison of IMS data demonstrated that isobaric asphaltenes from light and heavy oils shared similar collisional cross sections (CCS) with different homologous series in the experiments. Two-dimensional (2D) difference mobility plots showed that minor compounds in these fractions possess different CCS. The combination of CCS data with UHR MS and Py-GC-HR MS still was not enough to indicate if there is a single asphaltene architecture in the studied samples. However, the obtained data were structurally slightly more consistent with the island architecture. Furthermore, the systematic combination of mass spectrometry techniques and calculations provided a reliable method for fingerprinting complex geological samples and providing structural information.