The uncontrolled proliferative capacity of tumour cells, a hallmark of cancer, has been the main focal point for imaging modalities that provide non-invasive and quantitative estimates of tumour growth. Over the past few decades, several tracers have been developed for use with positron emission tomography (PET) to assess cell proliferation. More commonly, the exploitation of thymidine kinase-1 (TK1) substrate [18F]-labeled-3-deoxy-3-fluorothymidine ([18F]-FLT) uptake for imaging of proliferation has been broadly accepted but, its limitation in accurately depicting the S-phase fraction has become more apparent over the past 10 years. This study explores the use of deoxythymidylate kinase (dTYMK) as a plausible imaging and therapeutic target since dTYMK participates in the only known pathway to synthesise deoxythymidine diphosphate and ultimately deoxythymidine triphosphate (dTTP). We introduce the first use of a novel squaramide-nucleotide radiotracer combined with the sensitivity of PET imaging to trace the proliferative tumour fraction with respect to the convergent enzyme, dTYMK. Initial in vitro [18F]-SqFLT uptake in salvage proficient (HCT-116), de novo proficient (OST TK1) and CRISPR/Cas9 edited dTYMK knockdowns (B1 and B5) was found to be significantly low (~0.2 % ID/mg protein) when compared to [18F]-FLT (~20 % ID/mg protein) suggesting that, [18F]-SqFLT is not a substrate for dTYMK and, its rate-limiting step may be due to a low passive diffusion. As a pilot study, our observations were extended into an in vivo setting, which revealed non-significant tumour uptake in both wild-type and dTYMK knockdown models when compared to muscle. The highest accumulation of [18F]-SqFLT occurred in the kidney, liver and bladder. A high uptake was also observed in the gall bladder indicating partial excretion via the biliary pathway. While [18F]-SqFLT was unsuccessful in tracing the tumour proliferative fraction, the study still provided pharmacodynamic information into the increasing interest of nucleoside analogues, presenting squaramide phosphate mimics, as potential biologically active cancer agents. Moreover, the CRISPR/Cas9 edited dTYMK knockdown models served as a good platform for understanding some of the mechanisms that may account for dTYMK targeted radiotracer accumulation and retention in cells. A key finding in this study was the disparity between in vitro and in vivo growth rate of dTYMK knockdown models. It was concluded that a dTYMK bypass mechanism that becomes more apparent in vitro than in vivo, may exist to sustain DNA synthesis and maintain genomic integrity. Given the increasing interest in targeting dTYMK as part of an adjunct therapy, these models present as a good system for future pharmaceutical application. To conclude, the exploitation of dTYMK from an imaging endpoint remains challenging; however, success will allow detailed evaluation of the cellular metrics of proliferation and overcome the key limitations associated with [18F]-FLT imaging.