Besides the importance in drug design, recombinant nucleoside and nucleotide kinases are valuable biocatalysts to produce modified nucleotides in enzymatic in vitro processes. Enzymatic approaches offer an interesting alternative to the chemical synthesis by the use of mild reaction conditions, having a high regio-and stereoselectivity and by avoiding protection and deprotection steps. [13,14] Due to the importance of nucleoside and nucleotide kinases the characterization of their substrate spectra and the identification of the most efficient phosphate donor is highly relevant. Therefore, different methods were developed to determine kinase activity. Assays applicable towards a wide range of natural and modified substrates and phosphate donors include radiometric and chromatographic (TLC, HPLC) methods. But these assays are time-consuming and hardly suitable for high throughput applications. In addition, they would have to be optimized for some substrates. Other assays focus on the detection of adenosine 5'-triphosphate (ATP) consumption or production, since ATP is the most commonly used phosphate donor for various kinases. [16] Assays based on lactate dehydrogenase, pyruvate oxidase, malachite green, pyruvate kinase, fluorescence polarization, FRET or ELISA use the principle of measuring colorimetric or fluorometric changes. [17,18] Although these assays offer the opportunity to be used in high throughput, they are often expensive (especially if an antibody is involved), and require multi-step enzyme cascades. While the latter is not a concern when studying protein kinases, it is challenging for reactions catalyzed by nucleoside or nucleotide kinases because there is a higher risk that the non-natural nucleotides interfere with ATP detection. [17,18] In contrast, bioluminescence-based assays using a luciferase to detect ATP are fast, high throughput compatible and based on only one enzyme. In these assays kinase activity is determined in two successive reactions. First, the kinase of interest catalyzes the transfer of a phosphate from a phosphate donor (mainly ATP) to the respective substrate. Afterwards, the residual phosphate donor is converted into a luminescence signal by a luciferase (Scheme 1). Thus, the kinase activity inversely corresponds to the residual phosphate donor concentration. Due to the advantages, bioluminescence-based assays have already been applied successfully for the activity detection as well as for inhibition and kinetic studies of many kinases ,