Myt1 is an important cell cycle regulating kinase. Herein, systematic substrate studies revealed the kinase to be restrictive in terms of substrate acceptance. While simplified proteins (peptides) were not accepted, full-length proteins were efficiently phosphorylated. Since protein substrates are problematic in screening assays, a kinase binding assay based on time-resolved fluorescence resonance energy transfer was established and a first inhibition profile for this kinase was obtained. As an alternate assay, a fluorescence polarization based kinase binding assay was developed and formerly unknown inhibitors were identified. Finally, peptide microarray studies helped resolve the Myt1 substrate issue. Indeed, two peptides derived from this approach could be verified and validated as Myt1 substrates also in solution phase.