Here, we present the development of a straightforward methodology to regioselectively phosphorylate the 5′-OH group of unprotected nucleosides. We employ cyclosaligenyl phosphoramidite reagents together with pyridinium trifluoroacetate as activator, followed by in situ oxidation to prepare a panel of novel nucleoside-based chemical probes, ProLabel compounds 4–15. Alternative procedures for this transformation are available, but are limited in number and scope. Furthermore, the benefits of the new methodology include milder reaction conditions, wider solvent applicability, and, by avoiding sensitive reagents, a more straightforward handling of reagents, reactions, and workup processes. The panel of novel cyclosaligenyl phosphotriester uridine ProLabels have variable 2′ substituents (H, F, Cl, Br, I) as well as four different cyclosaligenyl groups that would span a range of half-lives for in vitro applications.