Abstract: The influences of ionization sources, solvents and mobile phases on the ionization of petroleum acids were studied. Negative-ion ESI using methanol as solvent and mobile phase yields the cleanest spectra with good sensitivity among the ionization techniques were evaluated. The composition of petroleum acids of two diesels were analyzed by established method. The results show that the compositions of petroleum acids in two diesels are similar. Besides the abundant amount in naphthenic acids, some aromatic acids and aliphatic acids are also discovered. A scientific and efficient method will be supplied for the further research in petroleum acids.