Overview of screening assays used to evaluate PROTACs for cancer treatment.

Abstract

Proteolysis-targeting chimeras (PROTACs) are promising anticancer agents, but optimizing them requires screening strategies that go beyond simple target-degradation readouts. This review provides an overview of assays for evaluating anticancer PROTACs and classifies them by their role in the degradation pathway, including target binding and engagement, ternary complex formation, ubiquitination, target degradation and evaluation of anticancer activity in vitro and in vivo. The literature (articles published from 2020 to 2025) shows a strong dependence on degradation endpoints, especially western blotting, and relatively little use of direct ubiquitination assays, ternary complex measurements and quantitative kinetic methods. We highlight knowledge gaps and propose a staged screening framework that integrates mechanistic, quantitative, phenotypic and translational assays to advance the discovery of anticancer PROTACs.