Standard toxicological in vivo testing has been challenged as the procedures are time-consuming, expensive, and require a large number of animals; given the number of problematic chemicals. Novel toxicological frameworks, such as "toxicity testing in the 21st century", proposed the use of "new approach methods" (in vitro and in silico techniques), that can be applied in high-throughput setups and would allow for the testing of a large number of compounds. However, such new approach methods need to be designed and evaluated first. Especially within ecotoxicology, the coverage of species-specific bioanalytical tools, e.g. for fish, is rather scarce. Currently, mainly in vitro assays of mammalian and bacterial origin are used. This thesis outlines how to design and scrutinise fish transient reporter gene assays. We have established transient reporter gene assays in permanent zebrafish fibroblasts and hepatocytes of the oxidative stress response and the xenobiotic metabolism toxicity pathways. We identified non-specific effects caused by transient transfection itself and suggested preventive strategies. Further, we identified toxicity pathways’ cross-talk as a significant driver of uncertainty in regards to the assessment of receptor-mediated toxicity. Additionally, we evaluated the correlation between cytotoxicity in cultured zebrafish cells and the acute toxicity observed in zebrafish embryos. When using chemical distribution models to derive bioavailable concentrations, we observed a good positive correlation between the two test systems. The results advocate an intensified use of fish in vitro assays in integrated testing strategies. Conclusively, new approach methods, as developed and applied in this thesis, show great potential in future toxicity testing and environmental monitoring.