Nucleoside 5'-triphosphate (NTP) analogues are valuable tools for biochemical and medicinal research. Therefore, a facile and efficient synthesis of NTP analogues is required. Here, we report on an improved nucleoside 5'-triphosphorylation procedure to obtain pure products after liquid chromotagrpahy (LC) separation with no need for high performance liquid chromatography (HPLC) purification. To improve the selectivity of the reaction we attempted the optimization of several parameters such as solvent, pyrophosphate nucleophilicity, time and temperature of the reaction. Eventually, the reaction was optimized by decreasing the temperature to -15 degrees C and increasing the reaction time to 2 hours, based on monitoring time-dependent product distribution using (31)P NMR. Furthermore, the NTPs were obtained as pure products after LC separation, which was impossible in the original Ludwig procedure. Good yields were obtained for all studied natural and synthetic nucleosides.