The endonuclease activity of influenza A virus RNA polymerase allows the digestion of host mRNA. The PA endonuclease domain could be a target of anti-influenza A virus drugs such as glycoconjugates. To test this activity, purified viral PA endonuclease domain protein is necessary. Here, we describe a method for the expression and purification of recombinant influenza A virus PA endonuclease domain protein, and a PA endonuclease assay to test glycoconjugates for potential inhibitory activity. Using influenza A virus PA cDNA as a template, DNA from the open reading frame of the PA endonuclease domain protein was amplified with PCR and cloned into an expression vector. Six His-tagged PA endonuclease domain proteins were expressed in Escherichia coli and purified with Ni2+-agarose resin and imidazole using an ion-exchange column. Using the recombinant PA endonuclease domain protein, an endonuclease assay was performed.