Protein kinases constitute a large enzyme family with key roles in cellular signal transduction. One way to elucidate the functions of protein kinases is to systematically identify their downstream targets. We present here a simple and effective method to identify direct protein kinase substrates in native cell lysates. First, we isolate the activity of the kinase of interest by engineering the normal kinase to utilize bulky ATP analogs that cannot be used by normal cellular kinases. This allows specific labeling of substrates with thiophosphate tags by performing kinase reactions in cell lysates that also include bulky ATP-γ-S analogs. After digesting the proteins in the reaction mixture, thiophosphopeptides are isolated using a single-step capture-and-release protocol and identified by mass spectrometry. This technique is easy to use and generally applicable.