<p>Border cell migration in the Drosophila ovary has emerged as a genetically tractable model for studying collective cell movement. Over many years border cell migration was exclusively studied in fixed samples due to the inability to culture stage 9 egg chambers in vitro. Although culturing late-stage egg chambers was long feasible, stage 9 egg chambers survived only briefly outside the female body. We identified culture conditions that support stage 9 egg chamber development and sustain complete migration of border cells ex vivo. This protocol enables one to compare the dynamics of egg chamber development in wild-type and mutant egg chambers using time-lapse microscopy and taking advantage of a multiposition microscope with a motorized imaging stage. In addition, this protocol has been successfully used in combination with fluorescence resonance energy transfer biosensors, photo-activatable proteins, and pharmacological agents and can be used with wide-field or confocal microscopes in either an upright or an inverted configuration.</p>