Recent literature has demonstrated the sensitivity of mayflies to environmental contaminants. However, to date, there are no methods approved by the US Environmental Protection Agency for using sensitive insects like mayflies in whole-effluent toxicity or receiving water toxicity tests. The parthenogenetic mayfly Neocloeon triangulifer has been shown to be amenable to continuous culture in the laboratory, and methods have been described for its use in both acute and chronic toxicity studies. The goal of the present study was to investigate aspects of N. triangulifer testing and culturing methods that might require adjustment so that they are applicable for testing effluents and receiving waters in a short-term exposure. To this end, the influence of organism age, test duration, and test temperature on sensitivity to NaCl as a reference toxicant were tested (concentrations ranging from 182 to 2489 mg/L). Further studies were conducted to assess the utility of commercially available diets and the influence of nutrient amendment of water on organism growth and sensitivity. Seven-day NaCl tests started with less than 24-h-old larvae were similar in sensitivity to 14-day and full life chronic tests, and were much more sensitive than those started with 7-day-old organisms. Reducing test temperature from 25 °C to 22 °C had a minor influence on culture timing, and little impact on sensitivity to NaCl. In other experiments, reconstituted test water supplemented with nutrients to potentially improve in-test food quality had minimal effect on growth at 7 days and did not significantly alter acute sensitivity to NaCl relative to unamended reconstituted water. A suitable commercially available, ready-to-feed diet substitute for cultured diatoms was not found. Testing N. triangulifer in effluents or receiving waters with the methods recommended will complement similar methods for Ceriodaphnia dubia.