Fear-potentiated startle (FPS) and prepulse inhibition (PPI) of acoustic startle are two widely used paradigms specifically designed to capture the impact of negative emotion (e.g. fear) and preattentive function on startle response. Currently, there is no single paradigm that incorporates both FPS and PPI, making it impossible to examine the potential interactions between fear and attention in the regulation of startle response. In this study, we developed an integrated FPS and PPI test protocol and validated it with psychoactive drugs. In Experiment 1, male Sprague-Dawley rats were randomly assigned to one of five groups, receiving either Light -Shock conditioning trials, non-overlapping Lights and Shocks, Light alone, Shock alone, or no Light and Shock. They were then tested for startle response and PPI concurrently, under the Light or No Light. FPS was observed only in rats subjected to fear conditioning, whereas all rats showed PPI and startle habituation. Experiment 2 used this paradigm and demonstrated a dissociative effect between diazepam (an anxiolytic drug) and phencyclidine (a nonselective NMDA receptor antagonist) on FPS and PPI. Diazepam suppressed both FPS and PPI, while PCP selectively disrupted PPI but not FPS. The diazepam’s anxiolytic effect on FPS was further confirmed in the elevated plus maze test. Together, our findings indicate that our paradigm combines FPS and PPI into a single paradigm, and that is useful to examine potential interactions between multiple psychological processes, to identify the common neural substrates and to screen new drugs with multiple psychoactive effects.