The morphogenic signal Shh provides in the developing CNS induces proliferation of neuronal precursor cells in the developing cerebellum and other tissues. Proliferative signaling by Shh is involved in the development of cancer, including specific brain and skin cancers such as basal cell carcinomas. Signaling takes place through a Patched (PTC-1)⁄Smoothened (SMO) Receptor complex. The activation of Patched by Shh releases the inhibition of Patched on Smoothened leading to Sonic Hedgehog pathway activation of the Gli transcription factor to induce downstream gene expression. The CellSensor® Gli-bla 22Rv1 cell line contains a beta-lactamase reporter gene under control of the Gli response element stably integrated into 22Rv1 cells. This cell line is a clonal population isolated by flow cytometry. The clone was selected based on inhibition of the pathway with KAAD-Cyclopamine. This cell line has also been tested for assay performance under variable conditions, including DMSO concentration, cell number, compound incubation time, and substrate loading time and validated for Z’ under maximal inhibition of β-lactamase activity with Clavulanic Acid.