Novel starting points for medicinal chemistry programmes can be effectively identified by screening libraries of fragment molecules in biochemical assays at high concentration. The key to success with this approach is the combination of a high quality fragment library with sensitive biochemical screening methods. There are an increasing number of literature reports where weakly active fragment molecules have been identified by high concentration biochemical assays. We have successfully demonstrated the use of high concentration screening of fragments, using a portfolio of single-molecule Fluorescence Correlation Spectroscopy (FCS+plus) detection techniques to ensure the highest reproducibility and sensitivity, and have determined the binding mode of active fragments to target proteins by X-ray crystallography. Further biophysical detection methods are reviewed for their applicability to studies of fragment binding.