Progress from our present understanding of the mechanisms behind mitosis has been compromised by the fact that model systems that were ideal for molecular and genetic studies (such as yeasts, C. elegans, or Drosophila) were not suitable for intracellular micromanipulation. Unfortunately, those systems that were appropriate for micromanipulation (such as newt lung cells, PtK1 cells, or insect spermatocytes) are not amenable for molecular studies. We believe that we can significantly broaden this scenario by developing high-resolution live cell microscopy tools in a system where micromanipulation studies could be combined with modern gene-interference techniques. Here we describe a series of methodologies for the functional dissection of mitosis by the use of simultaneous live cell microscopy and state-of-the-art laser microsurgery, combined with RNA interference (RNAi) in Drosophila cell lines stably expressing fluorescent markers. This technological synergism allows the specific targeting and manipulation of several structural components of the mitotic apparatus in different genetic backgrounds, at the highest spatial and temporal resolution. Finally, we demonstrate the successful adaptation of agar overlay flattening techniques to human HeLa cells and discuss the advantages of its use for laser micromanipulation and molecular studies of mitosis in mammals.