As a member of the Wee-kinase family Myt1 kinase is involved in G2/M checkpoint regulation of the cell cycle. Recently, a peptide microarray approach led to the identification of a short-chain peptide EFS247-259 as substrate of Myt1 kinase, which allowed for subsequent development of an fluorescence polarization based activity assay. The developed Myt1 activity assay was used to investigate the influence of external factors and to characterize the Myt1 catalyzed phosphorylation of EFS247-259. For the first time the kinetic parameters Km, Km, ATP and vmax were determined. Additionally 932 compounds from different databases and the published protein kinase inhibitor sets (PKIS I and II), a set of small molecule ATP-competitive kinase inhibitors, were screened. So ten new Myt1 inhibitors were identified, providing three new scaffolds and allowed the first definition of structure-activity relationships for Myt1 inhibitors. The derived results provide the basis for further investigating the molecular mechanism of Myt1, the characterization of new enzyme inhibitors and lead structure optimization.