This non-radioactive kinase assay kit is a two-stage peptide phosphorylation/colorimetric detection assay kit. In the first stage, a biotinylated substrate peptide containing tandem repeats of Poly (Glu4-Tyr) is incubated with a tyrosine kinase enzyme sample in the presence of non-radioactive ATP and a Mn2+/Mg2+ co-factor cocktail. The kinase assay can be performed with substrate peptide either in solution, or bound to the well of a streptavidin-coated 96-well plate. The second stage involves the detection of phosphorylated substrate by Direct Enzyme Linked Immunosorbent Assay (ELISA) using a monoclonal anti-phosphotyrosine-HRP (Horse Radish Peroxidase) antibody conjugate. Tetramethylbenzidine (TMB) is provided as the HRP substrate.