A dramatic shift has occurred in how biologists use microscopy images. Whether experiments are small-scale or high-throughput, automatically quantifying biological properties in images is now widespread. We see yet another revolution under way: a transition towards using automated image analysis to not only identify phenotypes a biologist specifically seeks to measure ('screening') but also as an unbiased and sensitive tool to capture a wide variety of subtle features of cell (or organism) state ('profiling'). Mapping similarities among samples using image-based (morphological) profiling has tremendous potential to transform drug discovery, functional genomics, and basic biological research. Applications include target identification, lead hopping, library enrichment, functionally annotating genes/alleles, and identifying small molecule modulators of gene activity and disease-specific phenotypes.