Protein kinases are prime targets for drug development due to their involvement in various cancers. However, selective inhibition of kinases, while avoiding off-target effects remains a significant challenge for the development of protein kinase inhibitors. Activity-based protein profiling (ABPP), in combination with pan-kinase activity-based probes (ABPs) and mass spectrometry-based proteomics, enables the identification of kinase drug targets. Here, we extend existing ABPP strategies for kinase profiling with a site-specific analysis, allowing for protein kinase inhibitor target engagement profiling with amino acid specificity. The site-specific approach involves highly efficient enrichment of ABP-labeled peptides, resulting in a less complex peptide matrix, straightforward data analysis, and the screening of over ~100 kinase active sites in a single LC-MS run. The use of both trypsin and pepsin in parallel to generate the ABP-labeled peptides considerably expanded the coverage of kinases and exact binding sites. Using the site-specific strategy to examine the on- and off-targets of the Ephrin receptor (Eph) B4 inhibitor NVP-BHG712 showed binding to EphA2 with an IC50 of 17 nM (95% CI 10 - 28 nM) and EphB4 with an IC50 of 20.2 nM (95% CI 13.7 - 30.4 nM). Next to the known targets, EphA2 and EphB4, NVP-BHG712 bound to the discoidin domain-containing receptor 1 (DDR1) with an IC50 of 2.1 nM (95% CI 1.3 - 3.4 nM), suggesting that a DDR1-targeting regioisomer of NVP-BHG712 was analyzed. The promiscuity of XO44 toward ATP-binding pockets on other proteins facilitated the screening of an additional 475 sites, revealing inosine-5’-monophosphate dehydrogenase 2 (IMPDH2) as an off-target. Therefore, the presented approach, which can be fully automated with liquid handling platforms, provides a straightforward, valuable strategy for competitive site-specific kinase inhibitor target profiling.