This third edition expands on the previous editions by providing updated protocols and current methods that cover recent breakthroughs in Drosophila research. Chapters guide readers through FlyBase, versatile gene expression systems, analysis of microRNA function, single-cell transcriptome data and metabolism, recent applications of CRISPR for precise genome editing, transcriptional activation and cell lineage tracing, protein inhibition and tagging, optogenetic and optochemical control of tissue mechanics, AFM measurements, sample preparation for electron microscopy, live imaging of different tissues and organs, and quantitative image analysis. Written in the format of the highly successful Methods in Molecular Biology series, chapters include an introduction to their topic, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, as well as tips on troubleshooting and avoiding known pitfalls Authoritative and cutting-edge, Drosophila: Methods and Protocols, Third Edition serves as a useful and practical guide to new researchers and experts using Drosophila as a model system. Chapter 7 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.