Image-based high content screening is a powerful tool to gain systematic measurement of cell phenotype perturbation upon a given treatment. The combination of this technology with RNA interference, CRISPR-Cas9 and small molecules generates a comprehensive method to decipher and eventually tune complex biological processes, such as changes in cell morphology, cell and proliferation. This has been made possible by the availability of innovative robotics, imaging and computational systems, increasing the scale and the speed of the analysis. High content fluorescent microscopes allow to systematically assess the expression and the localization of multiple biomarkers, which can be imaged simultaneously using different wavelengths. The integration of high content imaging with automated image-analysis allows tracking multiple physical and logical parameters, thus generating a multi-dimensional picture of any biological process. I will describe several examples of image-based high content screenings, which allowed us to successfully identify functional hits starting from large collections of molecules of unknown function.