The development of fast microscopic imaging devices has enabled the application of automated fluorescence microscopy to pharmaceutical high-throughput drug-discovery assays, referred to as high-content screening (HCS). Initially, green fluorescent protein and its derivatives from Aequorea Victoria, and later anthozoan fluorescent proteins (FPs) have become potent tools as live-cell markers in HCS assays. We illustrate the broad applicability of classic and novel FPs to drug-discovery assays, giving example applications of the use of FPs in multiplexed imaging as fluorescent timers, photosensitizers and pulse-chase labels, and for robotically integrated compound testing.