We herein report the optimization and application of silver cationization (Ag⁺) in combination with laser desorption ionization (LDI) ultrahigh-resolution mass spectrometry (UHR-MS) to determine the structures of the sulfur-containing compounds present in heavy crude oil. A number of sulfur-containing model compounds were used to optimize the positive-ion mode LDI-MS conditions in the presence of a silver-complexing agent. Under the optimized LDI conditions, sulfur-rich heavy oil fractions were treated with the silver salt, where Ag⁺ coordinated with the sulfur atoms to speciate the sulfur species. The obtained results suggested that benzothiophenic, naphtheno-non-aromatic sulfides, and non-aromatic thiols were the major components present in the analyzed oil sample.