A rapid and quantitative measurement of protein kinase activity based on MALDI-TOF-MS was developed. Here, biotinylated substrate peptides with stable isotope labeling were used to enable the rapid and specific enrichment of the peptides. Protein kinase activities in two samples can be directly compared utlizing the mass-unit difference of the subsrate peptides with different isotope labels. We successfully evaluated the effect of an inhibitory drug on the cellular protein kinase acitivity.