Activity-based protein profiling is a powerful tool to study target engagement in complex biological environments. XO44 is a chemical probe based on the scaffold of a promiscuous kinase binder, which covalently reacts with a conserved lysine in the ATP-binding pocket of kinases. Pretreatment of cells with a drug (candidate) results in competition of XO44 binding, which can be interpreted as in situ target engagement. Here, a workflow is presented which is optimized for kinome coverage, reproducibility and data processing. This dataset pertains to the difference in kinome coverage when using different protocols and (strept)avidin beads.