Petroleum asphaltene is a complex natural organic mixture which plays a crucial role in the refining processes. In this work, asphaltenes derived from petroleum vacuum residue and its hydrotreated product were characterized by high-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Tandem mass spectra with narrow ion isolation window were realized by increasing the value of collision RF amplitude. Tandem mass spectra with 3 Da and 1 Da isolation windows were compared, which demonstrated the advantages of applying narrow isolation window of 1 Da. The tandem mass spectra of asphaltenes were simplified due to the narrow isolation window. Moreover, a better correlation between the parent and fragment ions obtained by narrow isolation window of 1 Da was suggested by moving the isolation window. Finally, aromatic hydrocarbons with more naphthenic rings were suggested for the molecular structures of the processed asphaltene according to the results.