The Drosophila S2 cell line is popularly used to study mitosis. In this cell line, multiple genes can be easily and efficiently knocked down by RNA interference (RNAi), and the associated mitotic phenotypes can be assessed with high-resolution microscopy after immunofluorescence or in a living cell. However, compared to untransformed cells in wild-type organisms such as yeasts or worms, mitosis in the S2 cell line is more variable and often looks abnormal even in RNAi-untreated cells. Therefore, in order to judge whether a phenotype is derived from RNAi of the target gene or is simply a variation of control cells, it is critical to prepare proper control samples and perform objective imaging and image analysis. Here, we discuss how bona fide mitotic phenotypes associated with RNAi can be identified, avoiding selecting false positives, in S2 cells.