Doxorubicin (DOX) is an effective chemotherapy drug for various types of cancer. However, acute and chronic cardiotoxicity of DOX hamper its clinical application. There is a great demand for the discovery of drugs against DOX-induced cardiotoxicity. This paper proposed a dual fluorescence cellular imaging assay for screening active compounds against DOX toxicity. Whole-well fluorescence images of cells in 96-well microplates were automatically acquired and reconstructed by using a fluorescence microscope coupled with a computer-controlled moving stage. DOX-injured cardiomyocytes were labeled with two fluorescent probes, namely, fluorescein diacetate and Hoechst 33342, to determine cell viability and apoptosis. The linear range and sensitivity of the proposed approach were evaluated and validated by a known active compound, rutin. The proposed approach was also successfully applied in screening active compounds from a clinically used herbal medicine ZhenQiFuZheng granule (ZQFZ), which consisted of two herbs, Astragalus membranaceus (Fisch.) Bunge and Ligustrum lucidum. Five active components were found and were further analyzed by liquid chromatography coupled with mass spectrometry. Hydroxytyrosol, neonuezhenide, salidroside, and cimidahurinine attenuated DOX-induced cardiotoxicity in a dose-dependent manner with EC₅₀ values of 198.0, 260.4, 621.7, and 45.79 μM, respectively. Western blot results indicated that these active compounds protected against cardiotoxicity by decreasing reactive oxygen species (ROS) accumulation and downregulating apoptosis-related Bax/Bcl-2 proteins. The proposed approach is efficient in screening active compounds from natural products and other complex mixtures.