Imazetapir is a herbicide used in soybean and corn crops worldwide. Ecotoxicological studies have shown that imazetapir promotes genotoxic, biochemical and individual effects in aquatic vertebrates. In this study, we evaluated the response of different biomarkers in adult specimens of Leptodactylus latinasus exposed under laboratory conditions to the imazetapir based-formulation Pivot® H (10.59% Imazetapir) mimicking two possible real acute scenarios. Both exposure scenarios considered were the runoff simulation (scenario1: 10 mg/L) and the direct spraying application (scenario2: 1000 mg/L).Different endpoints were evaluated at several ecotoxicological levels after 48 and 96 h of exposure including individual (biometric indices and behavior alterations), histological (liver pigmentation and tissue alterations), biochemical (catalase, glutathione system and cholinesterase activities) and genotoxic effects (induction of micronuclei and nuclear abnormalities). The exposure to Pivot® H during 48 h, induced inhibition of the glutathione-S-transferase activity in scenario1 and an increase of hepatic tissue alterations and acetyl-cholinesterase levels in scenario2. After 96 h, we demonstrated that imazetapir formulation induced a decrease in melanin and hemosiderin, an increase in catalase activity and induction of micronuclei in scenario1 while in scenario2 there was a decrease in the hepatosomatic index, and an increase in liver alterations and melanin reduction. The multivariate analysis allows to correlate biomarkers at the same level in exposed specimens. Accordingly, we conclude that populations of L. latinasus could be at risk after real scenarios of exposure to pesticides corroborating that the species is a good model for ecotoxicological studies in the region.