The fission yeast Schizosaccharomyces pombe is frequently used as a genetically manipulable model system, offering valuable understandings into cellular mechanisms. In the present study, a comprehensive step-by-step methodology for the research of the action mechanisms and detoxification by efflux pumps is showed. The protocol involves the thawing and culture of yeast cells in liquid medium under controlled conditions to ensure exponential growth. After that, a dose-response assessment is carried out by culturing wild-type cells in liquid medium, followed by exposure to increasing concentrations of the toxic substances. Optical density measurements are taken spectrophotometrically after exposure, and the process is repeated at least three times for quantitative analysis. Subsequently, defective mutants are selected to explore specific mechanisms of action or detoxification by efflux pumps, with cultures prepared and treated similarly to the wild type. Optical density measurements are again taken after exposure for quantitative analysis. This methodology ensures robust and reproducible results for the research toxic substances effects on S. pombe.-Schizosaccharomyces pombe is an adequate tool to evaluate contaminants toxicity.-Dose-responses curves are obtained on wild type to evaluate toxicity mechanisms.-This methodology ensures robust and reproducible results for the research toxic substances effects on S. pombe.