High content imaging and analysis offers a robust, high throughput method for analyzing large numbers of cells with the benefit of the spatial and temporal demarcation afforded by fluorescence microscopy. This presentation will provide an overview of high content imaging platforms, the fluorescent labels required to automate the segmentation of cells and their subcellular domains, and newly developed fluorescent assays. Emphasis will be placed on dyes that provide an indication of cell viability, mechanisms of cell death, proliferation and prelethal toxicity. Newly developed phenotypic assays—in particular those applicable to CRISPR-Cas9 mediated genome-editing—will be discussed. Novel probes and labeling approaches to report internalization of ligands and therapeutic antibodies will also be presented with reference to high content imaging.