We developed a motif-enriched phosphoproteome analysis workflow utilizing an in vitro kinase reaction to enrich a set of peptides with specific primary sequence motifs. The kinase reaction attached the phosphate tag to the peptides derived from phosphopeptide enrichment and subsequent dephosphorylation, facilitates the enrichment of the kinase substrates from phosphoproteomic samples. We applied this method to phosphoproteomic profiling of inhibitor treated cancer cells, and successfully profiled kinase inhibitors with different inhibitory spectra. We anticipate this workflow will be useful to target a specific set of the phosphoproteome with the wide variety of available recombinant protein kinases.