Background: The concept of targeted covalent inhibition and the impressive clinical success of ibrutinib (Imbruvica®), an irreversible inhibitor of Bruton’s Tyrosine Kinase (BTK) used in the treatment of B-cell malignancies, renewed the interest in irreversible kinase inhibitors throughout academic and industrial research. Methods for a suitable characterization of such inhibitors, aligned to their special requirements, still remain an underexplored area and usually require the labor-intensive synthesis of individually tailored probes. Methods: We established a chemoproteomic method based on the well-established Kinobead technology for analyzing unbiased target engagement of covalent inhibitors directly in living cells. The importance of assessing covalent inhibitors in a cellular context rather than in cell extract-based approaches was demonstrated by comparing the new cell-based Kinobead (cbKB) assay to a functional assay monitoring the effect of BTK inhibition on BCR signaling in a disease relevant cell system. Comparing selectivity profiles generated with the cbKB and a lysate-based assay (differential Kinobead profiling) enabled us to distinguish between covalent and non-covalent targets. For validating this approach we used high resolution mass spectrometry to identify reactive cysteines in unexpected target kinases. Results: We proofed the relevance of assessing covalent inhibitors in a physiological environment by comparing intracellular target engagement using the cbKB approach in intact primary B-cells with a standard lysate-based Kinobead assay. With the new cbKB assay we determined BTK engagement of different covalent BTK inhibitors correlating with the functional effect of BCR signaling inhibition. Due to a different environment for binding and reaction a lysate-based assay does not allow for the prediction of the functional effect. We determined intracellular kinase selectivity for a set of covalent inhibitors by mass spectrometry. By exploiting target dissociation effects of reversibly bound compound and non-equilibrium binding due to covalent reaction we distinguished irreversibly bound kinases from non-covalent binders for the tested inhibitors. Different selectivity profiles for clinically relevant compounds, all designed to covalently bind a homologous cysteine, were determined highlighting the potential of targeted covalent inhibition. The differential Kinobead profiling identified a kinase as covalent target of ibrutinib not containing a cysteine homologous to Cys481 in BTK. The specification of a distinct cysteine covalently modified by ibrutinib utilizing high resolution mass spectrometry validated this approach. Conclusion: With the cell-based Kinobead assay we established a method to investigate intracellular target engagement of covalent kinase inhibitors in a kinome-wide manner without the need for labor-intensive design and synthesis of suitable probes. Compared with cell extract-based assays, the new cbKB assay produces more predictive results for different covalent BTK inhibitors comparable to functional assays. This enables a possibility for assessing covalent target engagement for kinases a functional assay is not available. With our approach to distinguish reversible and irreversible (off-) target binding we can easily identify kinases offering suitable reactive cysteines in the ATP binding site for the potential development of covalent inhibitors. Using high resolution mass spectrometry analyses we are able to further specify the exact reactive amino acid covalently bound by the compound.