An acute toxicity study assessed the LC50 values for eight different amino acid ionic liquids (AAILs), featuring two cations, tetrabutylphosphonium P4444 and tetrabutylammonium N4444, coupled with four anions PHE, ASP, SER, and GLY. According to the OECD 203 standard for acute fish toxicity tests with guppy fish (Poecilia reticulata, all the AAILs exhibited low toxicity levels, and were practically nontoxic and harmless. The LC50 values surpassed 100 mg/L and 1000 mg/L. This study provides valuable insights for industrial professionals in utilizing tetrabutylphosphonium-based amino acid ionic liquids P4444 AA and tetrabutylammonium-based amino acid ionic liquids N4444AA in chemical processes, indicating their safety in aquatic environments. These promising results highlight the potential of incorporating these AAILs into diverse chemical processes while ensuring minimal ecological impact. © 2024 The Authors