Mizoribine (MZR), an immunosuppressive drug, has been used for treatment in organ transplantation, lupus nephritis and rheumatoid arthritis. Orally administrated MZR is adsorbed, phosphorylated to MZR 5'-mono phosphate (MZR-P) and then inhibit inosine 5'-mono phosphate dehydrogenase. In this way, MZR-P blocks nucleic acid synthesis, which in turn inhibits proliferation of T and B cells. In order to refine optimum individual dosage of MZR, it is important to measure MZR levels in serum. Although only available method is high-performance liquid chromatography analysis, it is not suitable for high-throughput measurement of MZR levels in serum. Here, we identified a novel nucleoside kinase, which phosphorylates MZR, and developed an enzymatic method for measuring serum MZR levels. This enzymatic method can be applied to an automatic clinical analyzer for measuring a set of serum samples.