Advanced microscopy and corresponding image analysis have evolved in recent years as a compelling tool for studying molecular and morphological events in cells and tissues. Cell-based High-Content Screening (HCS) is an upcoming technique for the investigation of cellular processes and their alteration by multiple chemical or genetic perturbations. The analysis of the large amount of data generated in HCS experiments represents a significant challenge and is currently a bottleneck in many screening projects. This article reviews the different ways to analyse large sets of HCS data, including the questions that can be asked and the challenges in interpreting the measurements. The main data mining approaches used in HCS are image descriptors, computations, normalization, quality control methods and classification algorithms.