The current trend in high content screening (HCS) has been the utilization of more complex model systems that mimic both structural and functional properties of cells in vivo. Complex model systems have been the main motivation behind the development of imaging bioinformatics systems for phenotypic screening, with the main drivers for the utility of these biological systems being high fecundity and a short life span. Similarly, 3D cell culture models have emerged as effective systems to study tumor initiation, biological processes and reversion properties with respect to therapeutic targets. In contrast to 2D cell culture models, 3D cell culture models (i) have different patterns of development and can be functional, (ii) respond differently to therapeutic targets, and (iii) have different patterns of gene expression. Hence, the 3D cell culture model is a potential model system to bridge between the 2D model systems and the animal studies. In some systems, 3D colonies are imaged using elegant specialized microscopy and profiled using 2D image analysis and global intensity measurements. In this tutorial, we will show how the 3D cell culture systems can be screened when they are imaged in 3D volume using confocal microscopy.