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In this study, we propose a protocol to study anxiety-like behavior in larvae using the plus maze with ramp (PMR). In the PMR, anxious behavior is measured by the time spent on the flat arms relative to the ramped arms of the apparatus. In the first phase of the study, animals at 5, 14, and 21 days postfertilization (dpf) were exposed to the PMR at different water column heights and session times. It was observed that animals at 14 and 21 dpf were more sensitive to the PMR and exhibited obvious anxiety-like behavior compared with larvae at 5 dpf. In the second phase of the study, to assess the robustness of the type-anxious response exhibited by the larvae, animals at 5, 10, and 14 dpf treated with anxiolytic drugs were exposed to PMR. Alcohol exposure showed a bimodal effect for animals at 10 and 14 dpf, with an anxiolytic effect at intermediate doses, with more exploration of the apparatus and time in the ramp arms. Higher doses caused a reduction in locomotor activity characteristic of drunkenness. Exposure to clonazepam produced anxiolytic effects and a reduction in locomotor activity at the highest dose in 10 and 14 dpf animals. On the contrary, 5 dpf animals showed unexpected effects, which we believe to be related to their stage of development. The results indicate that the PMR is an effective tool for assessing anxiety-like behavior in zebrafish larvae and is sensitive to anxiolytic drugs. 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