Nucleoside analogues are widely used to treat cancer , viral and bacterial infections. However, nucleoside analogue drugs are usually active only in the nucleotide form. Therefore, the therapeutic efficacy largely depends on the intracellular phosphorylation of the nucleoside analogue to a 5'-triphosphate. In three consecutive steps nucleoside analogues are phosphorylated by (deoxy)nucleoside kinases ((d)NKs), (deoxy)nucleoside monophosphate kinases and nucleoside diphosphate kinases. Thus, to assess the therapeutic potential of new compounds in advance, a high-throughput assay for nucleoside and nucleotide kinases would be of great importance.