The natural fear reflex, characteristic of both animals and humans, is a short and intense defensive reaction in response to a strong and unexpected external stimulus. In experiments, this reflex is often triggered by a strong and short sound stimulus (acoustic flinch reflex – ASR). The amplitude of the ASR and the degree of its dependence on modulating factors, such as a previous weak stimulus (prepulse), serve as markers of the state of sensorimotor gating and are widely used in neuropsychiatry and neurophysiology to assess disorders of mental functions. The magnitude of the ASR amplitude is a critical evaluation factor, and the accuracy of its determination depends on taking into account many technical conditions: the design of the experimental installation, the type and location of the mechanical-electrical sensor, the method of digitization and presentation of primary data, the protocol of the experiment, etc. This issue presents a methodological development for measuring ASR and its prepulse inhibition (PPI), which includes an original working camera, hardware and software, as well as an optimal testing protocol. During validation the technique on a group of outbred rats (Wistar), it was found that (1) the technique allows us to assess the heterogeneity of the group by the amplitude of ASR and conduct appropriate phenotypic clustering, and (2) repeated, after 7 days, testing of ASR and PPI in the same animals does not violate their initial clustering and does not significantly changes the measured parameters. These observations allow us to consider the methodology applicable for sequential testing of one group of animals before and after any experimental exposure, taking into account the dependence of the result on the cluster membership of the subgroups determined during the first test.