Protein kinase inhibitors (PKI) evolved as promising drugs for multiple diseases. However, PKIs’ promiscuity causes polypharmacological effects challenging therapeutic development. We show that the polypharmacology of PKIs can be inferred from their impact on transcription factors (TF) lying at the apexes of signaling pathways. Using a high-content reporter assay, we evaluated TF activity profiles (TFAP) in cells treated with Akt, CDK, Aurora, Raf, MEK, and ERK inhibitors. In each case, we found kinase-specific consensus TFAP signatures signifying the on-target PKI activity. Remarkably, proximal kinases had the highest similarity values of their PKIs’ consensus signatures. Furthermore, we show that individual PKIs exhibited the consensus, “on-target signatures” within certain “specificity windows” and distinct “off-target signatures” at other concentrations. We also show that the off-target PKI signatures permit pinpointed the underlying biological effects. Therefore, the TFAP approach provides clear-cut quantitative metrics for assessing the dominant PKI activity and concentration ranges where the on-target activity dominates cell response. Thus, this effect-based approach illuminates PKI biology invisible to target-based techniques streamlining the selection of kinase chemical probes and PKI drug prioritization.