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 kinome coverage of XO44 in 19 human cell lines as determined by chemical proteomics.