The human genome encodes for over 600 kinases, which are important targets for drug discovery in many diseases, such as cancer and autoimmune disorders. The majority of kinase inhibitors target the conserved ATP-binding pocket, which contributes to the difficulty of developing selective inhibitors. A lack of selectivity may contribute to high drug candidate attrition rates due to an unfavorable off-target profile. To prevent unforeseen side effects, as well as to accurately link inhibitor function to pharmacological effects and phenotypic readouts, efficient methods for determining target engagement across the cellular kinome are necessary. One way to address this problem is to profile the interaction landscape of kinase inhibitors in living cells using the broad-spectrum kinase probe XO44. Here, we describe a detailed protocol of the CellEKT workflow using XO44 to profile kinase inhibitors in a full dose-response manner across 200+ kinases. The protocol allows to determine the EC50 of up to three kinase inhibitors in a cell line of interest in 4 days.