Abstract: The purpose of this article is to transform scientific research results into comprehensive experiments in chemical biology to explore the effects of cell models on cytotoxicity testing. Firstly, monolayer cell(2D) and three-dimensional(3D) cell culture models for HepG2 were established. Then, ATP bioluminescence assay was used to detect the cytotoxicity of Doxorubicin（DOX）in the two cell models. The results show that the toxicity of DOX on the 3D multicellular spheroids was much lower than that of 2D cells. Results from further study show that the cell uptake of DOX in HepG2 3D multicellular spheroids was obviously lower than that of 2D cells. This experiment embodies the integration of cutting-edge fields such as in vitro cell model construction, 3D cell viability detection, drug uptake and distribution analysis. Experiments help students understand the whole process of scientific research and improve their scientific literacy and practical ability.