The identification of the efficacy target(s) for hits from phenotypic compound screens remains a key step to progress compounds into drug development. In addition to efficacy targets, the characterization of epistatic proteins influencing compound activity often facilitates the elucidation of the underlying mechanism of action; and, further, early determination of off-targets that cause potentially unwanted secondary phenotypes helps in assessing potential liabilities. This short review discusses the most important technologies currently available for characterizing the direct and indirect target space of bioactive compounds following phenotypic screening. We present a comprehensive strategy employing complementary approaches to balance individual technology strengths and weaknesses.