The protozoan parasite Trypanosoma brucei is the causative agent of African sleeping sickness, and there is an urgent unmet need for improved treatments. Parasite protein kinases are attractive drug targets, provided that the host and parasite kinomes are sufficiently divergent to allow specific inhibition to be achieved. Current drug discovery efforts are hampered by the fact that comprehensive assay panels for parasite targets have not yet been developed. Here, we employ a kinase-focused chemoproteomics strategy that enables the simultaneous profiling of kinase inhibitor potencies against more than 50 endogenously expressed T. brucei kinases in parasite cell extracts. The data reveal that T. brucei kinases are sensitive to typical kinase inhibitors with nanomolar potency and demonstrate the potential for the development of species-specific inhibitors.