During water treatment, antibiotics can undergo abiotic and biotic degradation processes, leading to the generation of known and unknown transformation products (TPs), which are sometimes more persistent and/or toxic than the parent compounds. Therefore, advanced analytical strategies are necessary to identify and assess their potential environmental risks. With this aim, non-target and suspect screening methodologies for the chemical analysis of antibiotic TPs can be combined with effect-based strategies (in silico and in vivo), computational evaluation, and the so-called “effect-directed analysis” (EDA). In this chapter, a variety of studies combining both approaches (chemical and effect based) to comprehensively assess antibiotic TP formation in diverse water treatment scenarios are compiled and discussed.