Background Microfluidic devices are being used for phenotypic screening of zebrafish larvae in fundamental and pre‐clinical research. A challenge for the broad use of these microfluidic devices is their low throughput, especially in behavioral assays. Previously, we introduced the tail locomotion of a semi‐mobile zebrafish larva evoked on‐demand with electric signal in a microfluidic device. Here, we report the lessons learned for increasing the number of specimens from one to four larvae in this device. Methods and Results Multiple parameters including loading and testing time per fish and loading and orientation efficiencies were refined to optimize the performance of modified designs. Flow and electric field simulations within the final device provided insight into the flow behavior and functionality of traps when compared to previous single‐larva devices. Outcomes led to a new design which decreased the testing time per larva by ≈60%. Further, loading and orientation efficiencies increased by more than 80%. Critical behavioral parameters such as response duration and tail beat frequency were similar in both single and quadruple‐fish devices. Conclusion The developed microfluidic device has significant advantages for greater throughput and efficiency when behavioral phenotyping is required in various applications, including chemical testing in toxicology and gene screening. Graphical and Lay Summary Monitoring the behavior of multiple zebrafish larvae on‐demand and at the same time is important in biological investigations. A quadruple‐fish electro‐fluidic device for behavioral phenotyping of zebrafish larvae in response to electricity is provided in this paper. This methodology can be used in many assays involving zebrafish as a model organism for disease studies and drug screening.