Covalent inhibitors have proven to be very successful therapies for a wide range of indications. To ensure that the potential for non-specific reactivity is minimized, all new covalent inhibitors should be screened against both the intended target as well as potential off-target proteins. Bruton’s tyrosine kinase (Btk) is a member of the Tec family of kinases that is involved in B-lymphocyte development, differentiation, and B cell receptor signaling. CNX-774 is a potent, selective, and orally available small molecule inhibitor of Btk that forms a ligand-directed covalent bond with Cys-481, a non-conserved amino acid within the active site of the enzyme. In this study, we have assessed the off-target reactivity and specificity of CNX-774, employing a panel of HPLC and LC-MS based assays. In biochemical assays, CNX-774 has demonstrated potent inhibition of Btk with an IC50 of <1nM in a continuous-read assay (Omnia, Invitrogen). The covalent bonding of CNX-774 to Btk was confirmed by incubating recombinant Btk protein with a 10-fold molar excess of CNX-774 for 1 hour at room temperature and analysis by MALDI-TOF MS. A shift of protein mass corresponding to the molecular weight of CNX-774 confirmed the covalent bonding of CNX-774 to Btk. Digestion of the covalently bonded Btk with pepsin followed by MSMS analysis established the bonding of CNX-774 to Cys-481. Cellular potency as well as prolonged duration of action of CNX-774 was demonstrated in Ramos cells by using a biotinylated covalent probe that targets the same Cysteine residue as CNX-774. In order to assess the reactivity and potential off-target non-specific binding of CNX-774, it was tested in a panel of assays that included glutathione (GSH) reactivity, plasma protein bonding in an albumin-depleted human plasma preparation, and general extractability in whole blood. Conjugation of CNX-774 with GSH did not occur after incubation with 10 mM GSH at pH 7.4 for 4 hours at 37oc. Off-target protein reactivity was assessed by incubating CNX-774 with albumin-depleted human plasma at a final concentration of 0.1 mM for 1 hr at 37oC, followed by analysis by MALDI-TOF MS. No change was observed in the molecular mass of any protein in the compound-treated plasma protein sample demonstrating that CNX-774 does not bond covalently to any of the high- to mid-level abundance human plasma proteins. The potential covalent bonding of CNX-774 to off-target proteins in blood was determined by incubating CNX-774 in fresh rat or human whole blood at 37oC and monitoring the compound concentration over time by mass spectrometry. CNX-774 was found to be >90% extractable after 2 hrs of incubation in both rat and human whole blood. These results demonstrate that CNX-774 has potent inhibitory activity towards the intended target, Btk, while achieving remarkable specificity in a variety of assays designed to assess off-target reactivity towards abundant cellular thiols and blood proteins. These studies demonstrate the utility of intelligently designed in vitro assays to determine specificity and off-target reactivity of targeted covalent inhibitors at the discovery stage of the drug development process minimizing the potential for unintended non-specific reactivity.