The elucidation of the cellular efficacy target and mechanism of action of a screening hit remain key steps in phenotypic drug discovery. A large number of experimental and in silico approaches have been introduced to address these questions and are being discussed in this chapter with a focus on recent developments. In addition to practical considerations such as throughput and technological requirements, these approaches differ conceptually in the specific compound characteristic that they are focusing on, including physical and functional interactions, cellular response patterns as well as structural features. As a result, different approaches often provide complementary information and we describe a multipronged strategy that is frequently key to successful identification of the efficacy target but also other epistatic nodes and off-targets that together shape the overall cellular effect of a bioactive compound.