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The natural product withaferin A (WFA) is a potent angiogenesis inhibitor and it targets the ubiquitin–proteasome pathway in vascular endothelial cells. We generated a biotinylated affinity analog WFA-LC₂B for use as a probe to study angiogenesis. WFA-LC₂B inhibits angiogenic sprouting in vitro and it causes levels of ubiquitinated proteins to increase in tumor necrosis factor-α-treated human umbilical vein endothelial cells, confirming the retention of WFA’s biological activity. We show that WFA-LC₂B forms protein adducts in endothelial cells which are competed by free WFA in vivo. This WFA-LC₂B analog will be useful to isolate the biological target of WFA.