Drug resistance stemming from vascular abnormalization poses a significant hurdle in the realm of tumor therapy. However, vascular normalization strategies offer promising avenues for addressing this challenge. One notable obstacle in this pursuit is the absence of well-defined targets for vascular normalization. The activity-based protein profiling strategy (ABPP) leverages highly reactive probes with remarkable capabilities for delving into uncharted proteins within living organisms, which allows us to explore transient protein–protein interactions, identify proteins within complex systems, and pinpoint the binding sites of small-molecule–protein complexes. In this chapter, we describe the construction of ABPP probes based on vascular normalization active molecules identified in previous studies, screening for activity, and their application to the identification and binding site resolution of the new target of vascular normalization, EphrinB2. This protocol serves as a successful case study, demonstrating its potential in validating new targets for vascular normalization. We anticipate its wide-ranging applicability not only for validating other novel vascular normalization targets but also for elucidating and resolving unidentified intracellular target proteins linked to active molecules.