A dive into the new psychoactive substances: a review of the use of zebrafish (Danio rerio) as an in vivo model.

New psychoactive substances (NPS) are a broad category of drugs that pose public health risks and have poorly characterized pharmacological properties. Further studies are critical for risk assessment, toxicological profiling, and intoxication management. Human evaluations are limited by ethical and safety constraints, making animal models essential. Among in vivo models, zebrafish (Danio rerio) deserves emphasis in the study of NPS due to neurobehavioral, metabolic, and toxicological alterations. Considering some advantages, such as high genetic and neurochemical homology to humans, small body size, external fertilization, and embryo-larval transparency, zebrafish is well-suited for high-throughput screening of NPS. Their central nervous system shares key neurotransmitter pathways with mammals, supporting neurobehavioral assays. Also, a conserved cytochrome P450 system allows metabolism studies. Zebrafish have been successfully used to investigate NPS effects, with embryo-larval models offering practical advantages for acute toxicity and high-throughput behavioral screening. Adults, in turn, are more appropriate for complex behaviors and long-term or chronic exposure protocols. This review synthesizes for the first time behavior, toxicology, and metabolism data from zebrafish studies with a range of NPS classes within a unifying framework, to deliver a comprehensive understanding of their in vivo activity. By focusing on the translational value of zebrafish research, this review bridges experimental toxicology and clinical and forensic applications, thus filling a significant gap in current knowledge and demonstrating how zebrafish models can uniquely accelerate NPS toxicity profiling by providing mechanistic and behavioral information with high translational value.