Australians produce approximately 5 billion litres of wastewater daily, which contains a mixture of chemicals. Industries that discharge wastewater into rivers, estuaries and oceans are frequently required to conduct direct toxicity assessments (DTA) of their effluent as a condition in their wastewater release permits to ensure that there are no unacceptable toxicant risks to the receiving environments. DTA has traditionally been conducted using whole organism (in vivo) tests. However, whole organism DTA can be expensive, is typically limited to a small number of species and endpoints (mortality, development, growth and reproduction), and poses an important ethical dilemma associated with the use of animals. Cell-based (in vitro) bioassays can include a much wider range of toxicological endpoints, and provide ethical, cost effective and high throughput alternatives to in vivo bioassays. The Saving Nemo project was developed to provide the human wastewater industry with a suite of ethical and high throughput in vitro bioassays as an alternative to current whole organism testing to assess the toxicity risks from wastewater released into aquatic systems. However, prior to the inclusion of in vitro bioassays in regulatory frameworks, it is critical to establish their environmental relevance and technical suitability. A literature review was carried out to identify in vitro bioassays that can be used in DTA testing. There biologically relevant endpoints were related to responses at the molecular or cellular level (measurable in vitro), as well as adverse outcomes at the organism level, through toxicity pathways. Eleven endpoints were identified: mortality, growth, development, reproduction, endocrine disruption, xenobiotic metabolism, carcinogenicity, oxidative stress, inflammation, immunotoxicity and neurotoxicity. The literature review was also used to identify a list of suitable bioassays for each endpoint, based on assay maturity, cost, and sensitivity, for further use in the Saving Nemo project.