A new methodology for simple, rapid, high throughput biological testing of potentially hazardous chemical and environmental samples has been developed, which is based on measurement of oxygen consumption of aquatic test organisms using phosphorescent oxygen-sensitive probes and detection on a fluorescent plate reader. Test organisms are exposed to potential toxicants and then allowed to respire in a sealed measurement compartment in the presence of soluble oxygen probe added to the sample. The resultant depletion of the dissolved oxygen causes an increase in sample fluorescence over time, thus reflecting the organism respiration rate and its alterations. Dedicated low-volume sealable 96-well plates provide improved sensitivity, convenience and miniaturization in such respirometric assays. These assays are carried out using standard laboratory tools and fluorescence plate readers, with multiple samples processed in parallel in 96-well microtitter plates. Oxygen consumption rate is a universal biomarker of general viability and metabolic responses of aerobic organisms, hence, this methodology is applicable to various organisms, including those currently used in toxicity testing. In this study, the new respirometric platform has been demonstrated with different organisms including prokaryotes (E.coli), eukaryotes (Jurkat T-cells), invertebrates (Artemia salina), and validated with toxicity testing of environmentally relevant chemicals. A panel of water samples discharged from wastewater treatment plants was also analyzed with this panel of test organisms. Organisms were exposed to the samples for a period of time (0–24 h depending on the organism chosen), and then assessed for their respiration rates (1 h assay). Toxicity was recorded as EC50, i.e., the concentration of the toxicant which caused a 50% decrease in the respiration compared to untreated organisms. Responses of various organisms to certain chemicals can be cross-compared and correlated to established toxicity tests (based on LD50). In terms of sample throughput, sensitivity, speed, flexibility and convenience, the new screening platform is seen as being superior to the existing toxicity tests currently used. It provides adequate assessment of biological hazards of complex chemical and environmental samples, allows for the monitoring of sub-lethal effects and provides information-rich data reflecting the mode of toxicity. It is therefore highly suitable for environmental monitoring and screening of potentially hazardous samples, including large scale programs such as EU Water Framework Directive and REACH.