While receptor binding is well recognized as a factor in influenza A virus (IAV) and coronavirus (CoV) host adaptation, the role of viral glycoprotein cleavage has not been studied in detail so far. Interestingly, recent studies suggest that host species may differ in their protease repertoire available for cleavage. Furthermore, it was shown for certain bat-derived CoVs that proteolytic activation provides a critical barrier to infect human cells. Understanding the role of glycoprotein cleavage in different species and how IAV and CoVs adapt to a new protease repertoire may allow evaluating the zoonotic potential and risk posed by these viruses. Here, we summarize the current knowledge on the emergence of a multibasic cleavage site in the glycoproteins of IAVs and CoVs in different host species. Additionally, we discuss the role of TMPRSS2 in virus activation and entry and a role of neuropilin-1 in acquisition of a multibasic cleavage site in different hosts.