Laser desorption mass spectrometry (LD-MS) was used to measure three asphaltenes, their sub-fractions separated using gel-permeation chromatography (GPC), and model compounds to ascertain their molecular weight distributions and averaged molecular weights. The asphaltenes were isolated from three crude oils: Maya (MY), Khafji (KF), and Iranian Light (IL). To optimize the measurement conditions for LD-MS, the effects of instrument mode, matrix, and laser energy were determined. Linear mode detection gave larger ion counts and higher signal-to-noise ratios than did reflector mode. The use of a matrix to determine the asphaltene molecular weight distribution was not useful as it enhanced the ionization of only some of the asphaltene fractions. The chosen laser energy provided significant ionization of the high-molecular-weight fractions, while minimizing polymerization and fragmentation. Various model compounds were measured to clarify the dependence of the ionization capacity on molecular structure. Pure and small alkyl-substituted aromatics ionize easily, and tend to polymerize at relatively low laser energies. Bridged aromatics are easily disrupted at the alkyl bridge and do not polymerize. Aliphatic hydrocarbons are very difficult to ionize in the absence of an appropriate matrix. The higher the molecular weight of a compound, the more difficult it is to ionize. The characterization of the asphaltenes was aided by separating each into six sub-fractions using GPC. These fractions fell into two groups: high- and low-aromaticity groups. In each group, the lower the molecular weight of a sub-fraction, the higher its aromaticity. Maya asphaltene (As-MY) had a larger molecular weight fraction than the other asphaltenes, but the distribution pattern of As-KF was similar to that of As-MY, except for this fraction. All the As-IL sub-fractions had lower molecular weights and higher aromaticity than the corresponding sub-fractions of the other asphaltenes. The averaged molecular weights of the asphaltenes were determined from the weighted average of the averaged molecular weight of each of the six sub-fractions. For As-MY, As-KF, and As-IL the values are 1657, 1628, and 1462 amu, respectively.