The measurement of the transcriptional activity of the HH signaling pathway is widely used as an indication of pathway activation. Luciferase reporter assays are powerful tools to measure the specific ability of a transcription factor to bind to its consensus sequence and to activate transcription of target genes. Here, we describe a protocol to measure the transcriptional activity of the HH pathway in normal and cancer cells. This technique allows studying the activity of GLI transcription factors and their modulation by drugs and/or other factors.