Drug resistance has emerged as a critical challenge in clinical cancer treatment. Here, we present a high-throughput screening protocol to identify therapeutic small-molecule inhibitors against drug-resistant cancer cells. We detail the steps for constructing drug-resistant cell models, executing the chemical screening process, and performing data analysis and validation. This protocol facilitates the rapid identification of therapeutic strategies for different types of drug-resistant cancers and aids in studying mechanisms. For complete details on the use and execution of this protocol, please refer to Zhang et al.