The analysis of live cells using automated fluorescence microscopy systems on an industrial scale is known as high content screening/analysis (HCS/A). Its development has been driven both by the demands of compound screening in the drug discovery industry and by the promise of whole genome functional analyses using siRNA knockouts. This chapter outlines the primary applications of HCS/A within the drug discovery process and in systems cell biology. It discusses specific issues which must be addressed when undertaking HCS/A, such as choice of cells, probes, labels, and assay type. Drawing from information gathered from surveys of key users of HCS/A in industry and academia, it then provides a detailed description of HCS/A user issues and requirements, before concluding with a summary of the imaging instrumentation currently available for live cell HCS/A.