Abstract: Electrospray ionization mass spectrometry (ESI-MS) and collisional activation technology were used to determine target compound and monitor key intermediates in peramivir synthesis, a novel anti-influenza virus agent. Chemical structure of impurity formed in the last step were identified to be dehydroxylation product of peramivir based on ESI-MS and MS². Dehydroxylation product of peramivir was origined the side reaction of hydrolization of intermediate (5).