In the last decade ethynyl-2’-deoxyuridine (EdU) treatment followed by click chemistry to a fluorescent azide has become the mainstay for biolabelling DNA synthesis in S-phase of the cell cycle. However EdU, as with other thymidine analogues, is polar and not likely to transverse cell membranes by passive diffusion. Consequently, EdU is likely reliant on nucleotide transporters to be transported across cell membranes. The possibility of different expression of transporters in a cell population or between different cell lines raises the question of whether a dose of EdU is uniformly accessible for DNA synthesis. To circumvent this we proposed applying the prodrug approach to biolabelling - this is coined the pro-label approach. We synthesised a panel of mono- and bis- acyl esters of EdU with variation of lipophilicity. We then confirmed the stability of the pro-label compounds in cell culture media and demonstrated they were substrates for esterases (leading to release of EdU). Next we evaluated the cytotoxicity and level of incorporation into cellular DNA of the pro-label compounds on two human cancer cell lines (MDA-MB-231 cells and PC-3 cells). We found that the cytotoxicity associated with extended compound exposure was reduced compared to EdU. The uniformity of cellular DNA labelling was increased with the pro-label compounds compared with EdU. Our findings show that the pro-label approach has been effective in improving the pharmacological parameters of an existing nucleotide DNA biolabel.