W e have designed and implemented a framework for creating a fully automated high-throughput phototransfection system. Integrated image processing, laser target position calculation, and stage movements show a throughput increase of O23Â over the current manual phototransfection method although the potential for even greater throughput improvements (O110Â) is described. A software tool for automated off-line singlecell morphological measurements, as well as real-time image segmentation analysis, has also been constructed and shown to be able to quantify changes in the cell before and after the process, successfully characterizing them, using metrics such as cell perimeter, area, major and minor axis length, and eccentricity values. ( JALA 2010;15:329-41) DJC is a recipient of a 2009 ALA Young Scientist Award from the IEEE Robotics and Automation Society (www.ieee-ras.org).