Ubiquitin (Ub) conjugation to a substrate protein is a widely used cellular mechanism for control of protein stability and function, modulation of signal transduction pathways and antiviral responses. Identification and characterization of ubiquitinated viral proteins is an important step in understanding novel mechanisms of viral protein regulation as well as elucidating cellular antiviral strategies. Here we describe a protocol to easily detect and characterize the ubiquitination status of a viral substrate protein expressed either during infection or ectopically expressed as a fusion with a biotinylatable epitope tag. This tag provides advantages over current immunoprecipitation techniques by making use of the extremely tight biotin-streptavidin interaction. We provide an example of this protocol using the nonstructural protein 5 (NS5) from Langat virus (LGTV), a member of the tick-borne encephalitis virus (TBEV) serocomplex within the Flavivirus genus. Using the protocols outlined here, we describe some of the pitfalls inherent in determination of Ub linkage and demonstrate that NS5 is modified by at least two distinct ubiquitination types, multiubiquitination and K48-linked polyubiquitin chains.