Drug Toxicity studies are primarily used to investigate specific adverse events and specific endpoints such as cancer, cardiotoxicity, and skin/eye irritation. Drug discovery is implied here to make a specialization drug of it. Patulin (PAT), a mycotoxin, is widespread in agricultural products. Although available research results suggest that PAT can be toxic to the nerves, immune system, and skin leading to heart, liver, and kidney damage. Trypan blue staining and Hoechst 33342 staining were used to analyze PAT, which induces apoptosis in 293 T cells. Superoxide dismutase (SOD), GSH, and malondialdehyde (MDA) were used to measure changes in the state Oxidative stress status of 293 T cells induced by PAT. Changes in reactive oxygen species (ROS) and ATP in mitochondria suggest a role for mitochondria when PAT induces cell damage and apoptosis. Through analysis of Cyt-C release assay, alteration of caspase activity, and correlation analysis, the potential mechanism of the mitochondrial apoptosis pathway was demonstrated. The results demonstrated that PAT induced cell damage significantly, and with increasing time and concentration, the cell survival rate decreased significantly. Hoechst 33342 staining and Trypan blue staining showed an increased rate of PAT-induced apoptosis. As PAT concentration increased, the activities of intracellular SOD and glutathione peroxidase decreased, and MDA content increased. Decreased intracellular ATP levels and accumulated ROS content suggest increased mitochondrial membrane permeability. Overexpression of Cyt-C triggered a cascade reaction of the enzyme caspase, leading to apoptosis. The results of the enzyme activity assay and correlation analysis indicated that caspase 3 was the most important in the cascade and was most correlated with caspase 8 and caspase 9. Drug discovery helps to make better medicinal components for this kind of effect.