Tools that can aid in vitro and in vivo imaging and also noninvasively determine half-life and biodistribution are required to advance clinical developments. A Function-Spacer-Lipid construct (FSL) incorporating fluorescein (FSL-FLRO4) was used to label vesicular stomatitis virus (VSV), measles virus MV–NIS (MV) and influenza virus (H1N1). The ability of FSL constructs to label these virions was established directly by FACScan of FSL-FLRO4 labeled VSV and MV, and indirectly following labeled H1N1 and MV binding to a cells. FSL-FLRO4 labeling of H1N1 was shown to maintain higher infectivity of the virus when compared with direct fluorescein virus labeling. A novel tyrosine 125 I radioiodinated FSL construct was synthesized (FSL- 125 I) from FSL-tyrosine. This was used to label VSV (VSV-FSL- 125 I), which was infused into the peritoneal cavity of laboratory mice. Bioscanning showed VSV-FSL- 125 I to localize in the liver, spleen and bloodstream in contrast to the free labels FSL- 125 I or 125 I, which localized predominantly in the liver and thyroid respectively. This is a proof-of-principle novel and rapid method for modifying virions and demonstrates the potential of FSL constructs to improve in vivo imaging of virions and noninvasively observe in vivo biodistribution.