The water flea has been used as a test organism of toxicity test for surface water. Toxicity test with water flea is categorized into two parts. One is acute toxicity test with observing immobility and mortality and the other is chronic toxicity test determined by survival and reproduction of water flea. Heartbeat measurement of water flea was designed as a short-term toxicity test in this study. Direct measurement of heartbeat under microscope by aid of video camera gives and early diagnosis of mortality in short time. Therefore, the effects of measuring illumination, measuring time, and non-feeding during the test on heartbeat of water flea was evaluated to establish a new test approach. Test organisms used in this study are Daphnia magna, a well standardizes toxicity test organism, and Simocephalus mixtus, a newly refined organism. IC50 values of these test organism by heartbeat measurement were compared and discussed. It was found that toxicity test by heartbeat measurement was a reproducible, easy and simple method accomplished in a few hours.