At the heart of cancer pathology lies the dysregulated cell cycle, which is often driven by aberrant activities of the cell cycle regulating, cyclin-dependent kinases (CDKs). Efforts to harness the therapeutic potential of modulating CDK activities have led to the development of inhibitors with tailored CDK selectivity. However, uniformity in the methods used to evaluate CDK inhibitor selectivity has been lacking and consequently, direct comparison and interpretation of selectivity profiles determined under different assay conditions is difficult. Determination of the inhibition modalities crucial to profiling selectivity of a CDK inhibitor requires thorough kinetic analysis carried out under comparable assay conditions. In this study, we employed a streamlined series of in vitro assays for profiling CDK inhibitors wherein intrinsic inhibition constants and cellular binding parameters were measured by using strategically designed enzymatic inhibition and complementary biophysical assays. Our findings demonstrate the effectiveness of this strategy in determining and quantitatively analyzing the selectivity and inhibition modality of a set of representative CDK inhibitors towards the major oncogenic, cell cycle CDKs. In addition, the assay results provide insights into the inhibitor-target interactions that extend beyond potency and selectivity.