The Hedgehog signaling pathway, first discovered in the fruit fly Drosophila melanogaster, was later found to be largely conserved in vertebrates and is crucial within diverse aspects of animal development, as well as for stem cells’ maintenance. Dysregulation of the pathway has been linked to various diseases,e.g. autoimmune disorders and cancer. Modern drug discovery aims to identify small molecule modulators of protein functions, because such molecules may become new drug candidates targeting cancers dependent on aberrant Hedgehog signaling.Prior to this study a high-throughput screen of a compound library consisting of 20,000 unique structures was performed. Assay conditions designed to e%amine Hedgehog activity reduction resulted in 96 compound hits. In the present study, these preliminary hits were re-examined by hit confirmation assays. Eight compounds were confirmed and chosen as hit compounds to be further examined to establish drug potential with respect to bioavailability factors. Investigation of chemical properties with respect to good druglikeness revealed that the eight compounds have the potential of becoming bioavailable drugs, although certain optimization steps may be desirable. Examination of biological effects with regard to compound selectivity revealed that compounds to some extent affected related signaling pathways such as Wnt and NF-êB. Compounds J1 and N5 were found to be false positives, inhibiting the Luciferase reaction, and were omitted from succeeding investigations. Among the remaining hits, compounds D3 and G4 were found to be involved in blocking protein transport, while the functions of compounds I3 and J3 were not clear from the experiments performed. Compound L1 was shown to reduce Gli2 levels in the nucleus, while M3 clearly disrupted tubulin structures, a key component of the cells’ cytoskeleton and cilia.