Small-molecule inhibitors of protein kinases attract widespread interest in the field of disease therapy because of their high specificity and ease of administration. However, dissecting the conformational inhibition dynamics of kinase inhibitors is still challenging. Here, simultaneously monitoring the conformational inhibition details and potency of Aurora A kinase inhibitors has been achieved by active isotope dimethyl labeling coupled with mass spectrometry-based quantitative lysine reactivity profiling. The conformational effects of inhibitors on lysine reactivity can be globally quantified to feasibly reveal the regions involved in the kinase dynamic inhibition. The half-maximum disturbance concentrations (DC50 values) of the conformation-specific lysine residues could directly represent the conformational selectivity and potency of kinase inhibitors. Further, K309 is discovered as a novel hotspot contributing to the inhibition of Aurora A kinase via the specific rotation of kinase activation loop. This quantitative lysine reactivity profiling strategy might greatly promote the development of targeted drugs.