Influenza D virus (IDV) is a new member of influenza virus that uses cattle as the primary reservoir and infects multiple agricultural animals. Similar to influenza C virus (ICV), IDV also has seven segments in its genome and has only one major surface glycoprotein, called the hemagglutinin-esterase-fusion (HEF) protein, for receptor-binding, receptor-destroying, and membrane fusion. HEF utilizes 9-O-acetylated sialic acids as its receptor and has both receptor binding and esterase activities, thus is a critical determinant of host tropism. Here, we summarize the methods to evaluate the glycan-binding and esterase activities of HEF in vitro. The glycan-bind property is monitored through glycan microarray, MDCK cell-binding assay, Hemagglutination assay, solid-phase lectin binding assay, and immunofluorescence of tissue sections, and its esterase property is analyzed via esterase enzymatic activity assay.