The aim of the present study was to evaluate the response in larvae of the anuran species Rhinella arenarum, Rhinella dorbignyi and Odontophrynus americanus exposed to glyphosate (GLY, 2.5mg L-1), cypermethrin (CYP, 0.013mg L-1), chlorpyrifos (CP, 0.1mg L-1) and glufosinate-ammonium (GLU, 15mg L-1) using two behavioral endpoints: mean speed (MS) and total distance moved (TD); and two enzymatic biomarkers: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). In order to assess a global response and to determine the most sensitive species, an integrated biomarker response (IBR) index was calculated. Behavioral biomarkers were tested at 1 and 60 min, and the enzymes at 60 min after exposure. The results showed that: (1) there were statistical differences between species in a series of responses in swimming behavior, and cholinesterase activities within the first-hour of exposure to CYP, GLY, and CP at environmentally relevant concentrations (ERC); (2) IBR determined that Rhinella species were the most sensitive of the species tested and (3) IBR provided a comprehensive assessment of the health status of species exposed to ERC of a wide variety of agrochemicals globally and frequently used.