The present paper describes the vibrational startle response assay (VSRA), a new robust, simple and automated in vivo medium- to high-throughput procedure for assessment of the escape response and its habituation in zebrafish larvae. Such behaviors enable fish larvae to escape from predator strikes in aquatic ecosystems. The assay is based on measuring the distance moved by each larva during the startle response evoked by repetitive vibrational stimuli. The iterative reduction observed in the response to a series of tapping stimulus in VSRA met the main criteria of habituation. Subsequently, the analysis of concordance using a battery of neuroactive compounds modulating different neurotransmitter systems demonstrated that the results of VSRA are highly predictive of the effects on other vertebrates. Finally, as a proof of concept, VSRA was used to test two relevant environmental pollutants at different concentrations. The results demonstrated that VSRA is suitable for concentration-response analysis of environmental pollutants, opening the possibility to determine the potency and the associated hazard of impaired escape response for the different compounds. Therefore, we suggest that VSRA could be a valuable tool for screening of chemical compounds capable of compromising predator avoidance behavior.