Imaging-based phenotypic screening uses cellular phenotypes to describe the drug performance, which generally focuses on single cellular feature, lacking of a comprehensive characterization. Here, we propose a high content phenotypic screening method based on expansion microscopy (ExM) assisted cell painting, which enables multi-channel imaging with approximately 50 nm resolution. As a demonstration, we applied this method to a phenotypic screening involving five drugs. The morphological attributes of three subcellular structures were summarized to consist a “fingerprint” describing the drug effect. The proposed method can provide comprehensive and detailed clues for drug evaluation, enriching the content of phenotypic screening.