The stalk of influenza neuraminidase (NA) has been a target of cleavage by various proteases, resulting in the release of catalytically active globular heads from virus particles. However, despite successful cases in a number of influenza subtypes, this strategy could not be applied to all influenza viruses due to high variation of the NA stalk. In the present study, reverse genetics was employed to construct non-pathogenic recombinant influenza A viruses, termed rgH1N1(LVPR) and rgH1N1(LVPR-GS), that harbor the NA of H5N1 virus engineered to contain a specific thrombin cleavage site at the stalk region. By using thrombin to cleave NA at its stalk, a productive extraction of NA globular heads could be obtained from purified rgH1N1(LVPR). Furthermore, it was found that the NA of rgH1N1(LVPR-GS) could be cleaved by endogenous thrombin present in embryonated chicken eggs, resulting in the release of NA globular heads into allantoic fluids. These data highlight the use of thrombin cleavage as an effective strategy for extraction of active NA heads directly from live viral particles not only of H5N1 but, theoretically, of any subtype of influenza A viruses.