The Hedgehog (HH) signalling pathway, a conserved means of cell-cell communication, is required for human development and for the maintenance and regeneration of many tissues. Dysfunctional HH signalling is associated with congenital malformations and with the growth of several cancer types. Most of the known pathway inhibitors target Smoothened (SMO), a GPCR-like protein and the main upstream activator of the HH pathway. Although targeting SMO has proven very promising, acquired resistance has compromised this therapeutic strategy. Therefore, there is a critical need to find and develop molecules that target different proteins in the HH pathway. In mammals, HH signaling relies on the primary cilium, a microtubule-based organelle important for many cellular signaling pathways. Therefore, we used NIH-3T3 mouse fibroblasts expressing Hh pathway-responsive GFP reporter and ARL13B, a cilium protein. This experimental design takes into account compounds acting on HH signaling and/or ciliogenesis by assessing transcriptional activation (GFP) levels and cilium morphology (mCherry), respectively. Our microscopy-based screen determined potential hits from a highly chemical diverse library of compounds. After confirmation and selection, we decided to investigate 30 compounds regarding their biological properties. Downstream assays are aimed at identifying the mechanism of action and the target. Ultimately, these small-molecules could open up new ways to interfere with HH signaling in disease.