The increasing need for ethical and efficient toxicological testing has driven the adoption of new approach methodologies (NAMs) to replace traditional animal models. Before NAMS became popular, animal testing was the primary method used to assess the dangers of chemicals and emerging pathogens. However, these studies are time extensive, expensive, and can lead to inconclusive results as these studies might not reveal the underlying physiological mechanism that the substance is affecting. This study aims to establish cell painting as a reliable new approach methodology to determine effects of chemical toxicity in cells. Cell painting highlights the different organelles and machinery of the cell by tagging the various parts of the cell with fluorescent dye. While this has been used to identify various parts within the cell, cell painting can also be used to observe organelle and mechanical changes. Using zebrafish cell lines as a model, this study aims to explore the potential of cell painting to enhance predictive toxicology and reduce reliance on mammalian models.