The hydrolysis of p-nitrophenyl acetate (p-NPA) catalyzed by seryl-histidine or histidine has been monitored by electrospray ionization mass spectrometry in the presence of the internal calibration, 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS). The half-life of p-NPA in the presence of 10 mmol·L -1 seryl-histidine or histidine at 25 ℃ was 370 min and 70 min respectively. With the occurrence of acetyl seryl-histidine and acetyl histidine in the reaction, and the fact that p-NPA was stable in the presence of 10 mmol·L -1 serine, an imidazolysis mechanism has been proposed, which is in accordance with the reported work.