The Victorian Centre for Functional Genomics (VCFG) provides biomedical researchers throughout Australia the ability to perform genome wide or custom boutique screens using array-based gene knockdown platforms targeting protein coding genes, microRNAs and long non coding RNAs. Library screening is performed in high throughput in 96 or 384 well format using automated liquid handling robotics, plate readers and/or automated microscopy. The VCFG has two high-end automated microscopes (Thermo Cellomics), which have multi-channel and multi-fluorophore detection capabilities as well as offering a live cell chamber and a robotic arm option for automated multi plate handling. The software algorithms for image capture and analysis are customisable and allow for unbiased, unsupervised clustering of image analysis from basic intensity readouts of whole or part(s) of cells to complex morphological phenotypes. This poster will describe the workflow for high content imaging used at the VCFG with examples of basic and highly sophisticated image quantitation and analysis. Unbiased imaging can be applied to a broad range of biological questions and can provide novel insights into the relationship of a cellular phenotype in response to a specific treatment.