Recently, protein kinases have emerged as some of the most promising drug targets, and therefore, pharmaceutical strategies have been developed to inhibit kinases in the treatment of a variety of diseases. CK2 is a serine/threonine protein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, as well as in tumorigenesis. Elevated CK2 activity has been established in a number of cancers, where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently, the development of CK2 inhibitors has been ongoing in pre-clinical studies, resulting in the generation of a number of CK2-directed compounds. In the present study, an unbiased evaluation of CK2 inhibitors TBB, TBBz and DMAT was carried out to elucidate the mechanism of action, as well as inhibitor specificity of these compounds. Utilizing a chemo-proteomic approach in conjunction with inhibitor-resistant mutant studies, CK2a and CK2a’ were identified as bona fide targets of TBB, TBBz and DMAT in cells. However, inhibitor-specific cellular effects were observed indicating that the structurally-related compounds had unique pharmacokinetic properties, suggesting differences in inhibitor specificity. Rescue experiments utilizing inhibitor-resistant CK2 mutants were unable to rescue apoptosis and cell cycle arrest associated with TBBz and DMAT treatment, suggesting the inhibitors had off-target effects. Exploitation of an unbiased chemo-proteomic approach revealed a number of putative off-target inhibitor interactions, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme Quinone Reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors, as well as drug specificity, which will assist in the development of more specific next-generation CK2 inhibitors.