The catalytic kinase activity of ZAP-70 is known to depend on the mode of phosphorylation of the two-tyrosine residues Y492 and Y493 in the so-called activation loop. Y493 phosphorylation leads to rearrangement of the activation loop, which frees up the catalytic center for further downstream signaling. On the other hand, Y492 phosphorylation has been invoked as a negative activity regulator. Indeed, phosphorylation on Y493 might follow by auto-phosphorylation of Y492. Having confirmed that phosphorylation of Y492 and Y493 can be selectively stimulated, we focused on cell lysates stimulated with anti-CD3 and anti-CD3 + H2O2 for the proteomics analysis.