Fluorescence live microscopy is a powerful technique to study complex cellular dynamics such as cell division. The availability of fluorescent markers based on GFP fusion proteins for virtually any cellular structure allows efficient visualization of specific processes, and the combination of different fluorophores can be used to study their coordination. In this chapter, we present methods for automated live cell microscopy to study mitotic gene function systematically and in high throughput. In particular, we provide protocols for efficient generation of fluorescent reporter cell lines stably expressing combinations of cellular markers, and provide detailed guidelines for optimizing imaging protocols for automated long-term live microscopy.