The small but mighty fruit fly Drosophila melanogaster presents an effective model system to peek into nature’s fundamental principles, with a long history of fruitful discoveries. It is especially true in Drosophila neuroscience, thanks to the powerful suite of genetic tools, the huge library of mutation/deficiency lines, the easy-to-access neuropils throughout the development process, the diverse and trackable behaviors, and the considerably small requirements in laboratory equipment. Researchers have been integrating advanced methodologies over the years to allow faster, higher-throughput, more accurate, and higher-dimensional data acquisition and analysis. Recent advances in optogenetics, high-throughput sequencing, multi-omics, imaging, machine learning-assisted detection and discoveries have significantly pushed the boundaries of the field on all fronts. It is truly an inspiring and critical time to work on the Drosophila nervous system. In this collection, we invite colleagues to contribute and document their state-of-the-art methodologies in Drosophila neuroscience. Utilizing JoVE’s unique and powerful platform, we hope to consolidate practical knowledge that might otherwise be lost in conventional reports. We look forward to taking this opportunity to disseminate ingenious methods fast and widely, accelerate neuroscience research in Drosophila, and translate philosophies to the broader field.