X-ray diffraction, liquid ¹H and solid ¹³C nuclear magnetic resonance (NMR), Raman, Fourier transform infrared (FTIR), laser desorption/ionization-time of flight (LDI-TOF) mass and fluorescence spectroscopy have been used to determine structural properties of asphaltenes obtained from Hassi Messaoud crude oil. While liquid ¹H- and ¹³C-NMR, IR, and LDI-TOF mass and fluorescence spectroscopy allow the determination of the chemical composition and structure of single asphaltene molecules, X-ray diffraction and Raman spectroscopy make it possible to asses parameters characterizing small asphaltene clusters. The proposed characterization of petroleum asphaltenes affords information that leads to better understanding of asphaltene flocculation conditions.