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A rapid toxicity test based on inhibition of esterase activity in the harmful freshwater microalgae –Microcystis aeruginosa was developed using flow cytometry. The hydrolysis rate of fluorescein diacetate (FDA) by intracellular esterase to fluorescein was used to indicate the metabolic activity of algae. Uptake of FDA was optimized at different concentrations and incubation times. Propidium iodide (PI) was utilized to assess cell membrane integrity. The optimized FDA/PI staining dosages were 10mg/L and 10μM, respectively, lower than the reported concentrations. Correspondingly, the proper incubation time was 14–21min at the optimal FDA dosage determined in this study. A new procedure based on optimized FDA/PI condition, called “whole algal culture flow cytometry with fluorescence triggering”, was developed for short-term bioassays. This new procedure, taking account of working conditions such as pH and …