Abstract Amphibia is the most threatened class among vertebrates, with >40% of the species threatened with extinction. Pollution is thought to alter amphibian population dynamics. With the growing interest in behavioral ecotoxicology, the neurotoxic organophosphate pesticides are of special concern. Understanding how exposure to neurotoxics leads to behavioral alterations is of crucial importance, and mechanistic endpoints should be included in ecotoxicological methods. In the present study, we tested an 8-day assay to evaluate the toxicity of two organophosphates, diazinon and chlorpyrifos, on Xenopus laevis, that is, on biochemical, morphological, and life-history traits related to locomotion capacities. The method involves measuring biomarkers such as glutathione-S-transferase (GST) and ethoxyresorufin-O-deethylase (EROD; two indicators of the detoxifying system) in the 8-day-old larvae as well as acetylcholinesterase (AChE) activity (involved in the nervous system) in 4-day-old embryos and 8-day-old larvae. Snout-to-vent length and snout-to-tail length of 4-day-old embryos and 8-day larvae were recorded as well as the corresponding growth rate. Fin and tail muscle widths were measured as well for testing changes in tail shape. Both tests showed effects of both organophosphates on AChE activity; however, no changes were observed in GST and EROD. Furthermore, exposure to chlorpyrifos demonstrated impacts on morphological and life-history traits, presaging alteration of locomotor traits. In addition, the results suggest a lower sensitivity to chlorpyrifos of 4-day-old embryos compared to 8-day-old larvae. Tests on other organophosphates are needed to test the validity of this method for the whole organophosphate group. Environ Toxicol Chem 2022;41:2688–2699. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.