Three-dimensional (3D) cell culture is emerging for drug design and drug screening. Skin toxicity is one of the most important assays for determining the toxicity of a compound before being used in skin application. Much work has been done to find an alternative assay without animal experiments. 3D cell culture is one of the methods that provides clinically relevant models with superior clinical translation compared to that of 2D cell culture. In this study, we developed a spheroid toxicity assay using keratinocyte HaCaT cells with propidium iodide and calcein AM. We also applied the transfer learning-containing convolutional neural network (CNN) to further determine spheroid cell death with fluorescence labeling. Our result shows that the morphologies of the spheroid are the key features in determining the apoptosis cell death of the HaCaT spheroid. Our CNN model provided good statistical measurement in terms of accuracy, precision, and recall in both validation and external test data sets. One can predict keratinocyte spheroid cell death if that spheroid image contains the fluorescence signals from propidium iodide and calcein AM. The CNN model can be accessed in the web application at https://qsarlabs.com/#spheroiddeath.