Discussion Identifying the targets of a bioactive compound is often the rate limiting step toward understanding the molecular mechanism of drug action. Current approaches rely on linking the bioactive compound to a surface or an affinity handle, permitting selective capture of interacting proteins for identification by mass spectrometry. A major consideration with these methods is insuring that the chemical derivatization of the bioactive compound does not disrupt the binding interactions with the cellular targets. We have developed a method based on a novel chloroalkane capture tag that minimally affects compound potency and cell permeability. This allows verification of the pharmacological activity of the modified compound, thus increasing the confidence in the biological relevance of captured proteins. In addition, by allowing the chloroalkane-modified compound to bind the targets within living cells, the cellular architecture during the binding step is preserved and better represents the conditions that the unaltered compound would normally engage these targets. Following binding with the tagged compound in live cells, the cells are lysed and the chloroalkylated compound and its associated targets are rapidly captured onto immobilized HaloTag protein and then released by competitive elution. The identified targets are then validated for direct binding relationship with the bioactive compound by bioluminescence energy transfer.