To help advance drug discovery projects, a new and validated search method is presented by which potential bioisosteric replacements can be retrieved from a database of more than 700,000 structural fragments. The heart of the search method is an optimized topological pharmacophore fingerprint which describes each fragment as a combination of attachment points, hydrogen bond donors and acceptors, hydrophobic centers, conjugated atoms, and non-hydrogen atoms. In the fingerprint the influence of the attachment point is enhanced by giving it extra weight relative to the other descriptors. The Euclidean distance has proven to be the optimum distance measure to compare the fingerprints in a database search. The performance of the pharmacophore fingerprint based search method has been validated using more than 2200 bioisosteric fragment pairs extracted in an unbiased procedure from the BIOSTER database. The true bioisosteric pairs have been compared with pairs of random fragments originating from the WDI database. Normalized by the standard deviation of the random pairs distance distributions, an excellent separation of true pairs from random pairs was obtained for R-group fragments (2.2 standard deviation units) as well as for linkers (2.6 units) and cores (2.6 units). The bioisoster search method has been implemented as an intranet application called IBIS and is now routinely used by Organon researchers.