Harmful algal blooms (HABs) that lead to mass mortality of aquacultured fish and shellfish are increasing in frequency, magnitude, and duration worldwide. Although a variety of hypotheses have been proposed for the mechanism behind the lethal effects of HAB species on fish and shelfish, the exact mechanism is still unclear. It is therefore essential to develop a method for rapid toxicity evaluation of HAB species collected in the field. In previous studies, the rotifer Brachionus plicatilis has been used in tests to evaluate the toxicity of HAB species; this test can quickly and easily evaluate toxicity at low cell densities. However, because the growth physiology of rotifers varies depending on the strain and culture conditions, it is necessary to establish rotifer test strains, culture conditions, and conditions for the toxicity test to make rotifer toxicity tests practical for use as a method to evaluate toxicity in the field. The aim of this study was to establish rotifer test strains, culture conditions, and conditions for the toxicity test. First, we investigated the effects of the dinoflagellate Heterocapsa circularisquama on four strains of rotifer (Brachionus rotundiformis, B. koreanus, and two strains of B. plicatilis). As a result, we selected B. plicatilis (Amami strain) for toxicity tests because it was the most sensitive to the toxicity of H. circularisquama. Second, we investigated the optimal temperatures and salinities for culture and toxicity tests in a growth experiment with B. plicatilis (Amami strain) under a crossed factorial design with 9 combinations of 3 temperatures (20, 25 and 30℃) and 3 salinities (25, 30 and 35). Our results indicate that toxicity tests should be conducted at water temperatures of 20–25℃ and salinities of 25–30. Furthermore, we applied the rotifer toxicity test using 8 species (10 strains) of cultured phytoplankton, including both HAB species and nontoxic species. The test detected the lethal effects of H. circularisquama, which has potent lethal effects on shellfish, and Karenia mikimotoi, which has potent lethal effects on fish and shellfish. However, the other phytoplankton species tested did not affect the percent survival of the rotifers, whether potentially toxic or not. Finally, we conducted rotifer toxicity tests using water samples from the field that contained H. circularisquama, Heterocapsa sp., and Chattonella antiqua; we succeeded in detecting only the toxicity of H. circularisquama. We conclude that the rotifer toxicity test using B. plicatilis (Amami strain) under optimum conditions is useful as a method for rapid toxicity evaluation of HAB species collected in the field, at least for those with potent lethal effects on shellfish, such as H. circularisquama and K. mikimotoi.