The resolution of cell-based assays down to the cellular level has created new opportunities for the drug discovery process. Aptly named high content analysis, such an approach is enabling new methods of analysis for the broad range of therapeutic targets emerging in the post-genomics era, and offering alternative multiparametric readouts for some traditional analyses. Microplate cytometry is one of the technologies that is being applied to a broad range of assays utilizing fluorescent labeling, at throughputs compatible with primary screening campaigns. Cellular resolution is achieved using scanning laser excitation coupled to photomultiplier detection. This configuration results in area-based scanning across a large field of view, plus simultaneous detection of up to four emission colors for efficient multiplexing. Microplate cytometry is being used most extensively in the field of oncology research because of its usefulness for numerous applications, including protein kinase activity, cell cycle analysis, and cell colony formation. The review focuses on the Acumen Explorer microplate cytometer (TTP LabTech Ltd., Melbourn, Hertfordshire, UK), detailing the principal components of the instrument and providing an overview of its use in high content screening.