The dinoflagellate Karenia mikimotoi, recognised as a harmful algal bloom (HAB) species, induces mass mortality among aquacultured fish and molluscan shellfish in coastal areas worldwide. Despite numerous studies examining the mechanism behind the lethal effects of K. mikimotoi on marine organisms, the exact toxicity mechanism remains unclear. Therefore, developing a method for readily assessing the toxicity of dinoflagellates collected from the field is crucial. While previous investigations have used the rotifer Brachionus plicatilis to evaluate the toxicity of HAB species, a comprehensive verification of K. mikimotoi, especially in its natural cell state, has not been conducted. Therefore, we aimed to demonstrate the efficacy of the rotifer toxicity test for K. mikimotoi. Initially, we investigated the toxic effects of three culture strains of K. mikimotoi (IMR4, KU9, and Km69-9ax) on juvenile abalones (Haliotis discus discus and H. gigantea) and the rotifer B. plicatilis. Exposure tests with juveniles of abalone (exposure density: 5.0–5.3×10⁴ cells mL^-1) and rotifers (exposure density: 10³ cells mL^-1) revealed that IMR4 significantly reduced the survival rate of juvenile H. discus discus (p<0.05) and H. gigantea (p<0.01), whereas KU9 and Km69-9ax exhibited no toxicity. The rotifer survival rate was lowest in the group exposed to the IMR4 strain (p<0.001), followed by those exposed to KU9 (p<0.001) and Km69-9ax strains (p<0.01). Rotifer survival decreased in an exposure density-dependent manner during IMR4 toxicity tests, and rotifers were killed even at a cell density of 10 cells mL^-1. Consequently, we classified the IMR4 strain as virulent and the KU9 and Km69-9ax strains as attenuated. Subsequently, no toxicity to rotifers was observed in groups exposed to the culture supernatant, ruptured cell suspension, heat-treated cell suspension, or non-contact conditions. However, the rotifer survival rate significantly decreased in the group exposed to the intact cell suspension of the IMR4 strain (p<0.001). Finally, we investigated the effects of natural seawater collected on 5^th and 6^th August, 2022 from the Yatsushiro Sea, which contained K. mikimotoi cells on H. discus discus and rotifers. Both juvenile H. discus discus and rotifers died rapidly after exposure to red-tide seawater of K. mikimotoi. Our findings indicate that the rotifer toxicity test can assess the toxicity of K. mikimotoi on mollusc shellfish with high sensitivity.