Discovering functional relationships of genes through cell-based phenotyping has become an important approach in functional genomics. High-throughput imaging offers the ability to quantitatively assess complex phenotypes after perturbation by RNA interference (RNAi). Such image-based high-throughput RNAi screening studies have facilitated the discovery of novel components of gene networks and their interactions. Images generated by automated microscopy are typically analyzed by extracting quantitative features of individual cells, resulting in large multidimensional data sets. Robust and sensitive methods to interpret these data sets and to derive biologically relevant information in a high-throughput and unbiased manner remain to be developed.