Molecular glue drugs exert pharmacological effects by inducing or stabilizing protein-protein interactions. Evaluating their activity not only requires assessing their binding to proteins, but also involves the evaluation of multiple aspects such as ternary complex formation, interface stability, cooperativity, and intracellular function. This review summarizes the commonly used methods and applications for evaluating the activity of molecular glues from three aspects: in vitro quantitative detection, cellular validation, and theoretical calculations and simulations; and proposes a hierarchical approach to activity evaluation based on the actual needs for efficiency in drug development, aiming to provide some reference for the study of molecular glue drug activity and the selection of evaluation methods.