Smoothened (Smo) is essential for transduction of the Hedgehog (Hh) signal in both insects and vertebrates. Binding of Hh to Ptc-Ihog relieves the Patched (Ptc)-mediated inhibition of Smo, which allows Smo to activate the cubitus interruptus (Ci)/Gli family of zinc fi nger transcription factors and thereby induce the expression of Hh target genes, such as decapentaplegic (dpp), ptc, and engrailed (en). The activation of Smo appears to be one of the most important events in Hh signaling. Studies have shown that Hh induces cell surface/ciliary accumulation and phosphorylation of Smo by multiple kinases, including protein kinase A (PKA), casein kinase 1 (CK1), casein kinase 2 (CK2), G protein-coupled receptor kinase 2 (Gprk2), and atypical PKC (aPKC). Here, we describe the assays used to examine the activity of Smo in Hh signaling, including in vitro kinase, ptc -luciferase reporter assay, cell surface accumulation assay, fl uorescence resonance energy transfer (FRET) assay, and wing disc immunostaining. These assays are powerful tools to study Smo phosphorylation and activation, which have provided mechanistic insight into a better understanding the mechanisms of Smo regulation.