Phosphoramidate pro-nucleotides (ProTides) have revolutionized the field of anti-viral and anti-cancer nucleoside therapy, overcoming the major limitations of nucleoside therapies and achieving clinical and commercial success. Despite the translation of ProTide technology into the clinic, there remain unresolved in vivo pharmacokinetic and pharmacodynamic questions. Positron Emission Tomography (PET) imaging using [18F]-labelled model ProTides could directly address key mechanistic questions and predict response to ProTide therapy. Here we report the first radiochemical synthesis of [18F]ProTides as novel probes for PET imaging. As a proof of concept, two chemically distinct radiolabelled ProTides have been synthesized as models of 3′- and 2′-fluorinated ProTides following different radiosynthetic approaches. The 3′-[18F]FLT ProTide was obtained via a late stage [18F]fluorination in radiochemical yields (RCY) of 15−30% (n = 5, decay-corrected from end of bombardment (EoB)), with high radiochemical purities (97%) and molar activities of 56 GBq/μmol (total synthesis time of 130 min.). The 2′-[18F]FIAU ProTide was obtained via an early stage [18F]fluorination approach with an RCY of 1−5% (n = 7, decay-corrected from EoB), with high radiochemical purities (98%) and molar activities of 53 GBq/μmol (total synthesis time of 240 min).