High-throughput screening (HTS) is a well-established process for lead discovery in Pharma and Biotech companies and is now also being used for basic and applied research in academia. It comprises the screening of large chemical libraries for activity against biological targets via the use of automation, miniaturized assays and large-scale data analysis. Since its first advent in the early to mid 1990s, the field of HTS has seen not only a continuous change in technology and processes, but also an adaptation to various needs in lead discovery. HTS has now evolved into a mature discipline that is a crucial source of chemical starting points for drug discovery. Whereas in previous years much emphasis has been put on a steady increase in screening capacity (‘quantitative increase’) via automation and miniaturization, the past years have seen a much greater emphasis on content and quality (‘qualitative increase’). Today, many experts in the field see HTS at a crossroad with the need to decide on either higher throughput/more experimentation or a greater focus on assays of greater physiological relevance, both of which may lead to higher productivity in pharmaceutical R&D. In this paper, we describe the development of HTS over the past decade and point out our own ideas for future directions of HTS in biomedical research. We predict that the trend toward further miniaturization will slow down with the balanced implementation of 384 well, 1536 well, and 384 low volume well plates. Furthermore, we envisage that there will be much more emphasis on rigorous assay and chemical characterization, particularly considering that novel and more difficult target classes will be pursued. In recent years we have witnessed a clear trend in the drug discovery community toward rigorous hit validation by the use of orthogonal readout technologies, label free and biophysical methodologies. We also see a trend toward a more flexible use of the various screening approaches in lead discovery, that is, the use of both full deck compound screening as well as the use of focused screening and iterative screening approaches. Moreover, we expect greater usage of target identification strategies downstream of phenotypic screening and the more effective implementation of affinity selection technologies as a result of advances in chemical diversity methodologies. We predict that, ultimately, each hit finding strategy will be much more project-related, tailor-made, and better integrated into the broader drug discovery efforts.