The Hedgehog pathway is a key regulator of embryonic patterning, tissue homeostasis, and stem-cell maintenance, and its inappropriate reactivation drives several human cancers. Signaling begins when a secreted Hedgehog ligand, such as Shh (Sonic Hedgehog) or Indian Hedgehog / IHH, binds the twelve-pass transmembrane receptor Patched1. In the absence of ligand, Patched1 catalytically suppresses the activity of the G-protein-coupled-receptor-like protein Smoothened; ligand binding relieves this inhibition, allowing Smoothened to signal within the primary cilium. The pathway output is determined by the GLI family of zinc-finger transcription factors. In the off state, GLI proteins, particularly Gli3 and to a lesser extent Gli2, are proteolytically processed into transcriptional repressors, a process dependent on the negative regulator SUFU, which sequesters GLI proteins, and on the kinesin KIF7, which organizes GLI processing at the ciliary tip. Pathway activation blocks this processing and promotes formation of full-length GLI activators, chiefly Gli2 and Gli1, with Gli1 itself being a direct transcriptional target that amplifies the signal and serves as a reliable readout of pathway activity. In cancer, constitutive Hedgehog signaling arises through loss-of-function mutations in Patched1 or SUFU and activating mutations in Smoothened, driving basal cell carcinoma and medulloblastoma, while ligand-dependent and stromal signaling contributes to many other tumors and to cancer stem-cell maintenance. Smoothened antagonists are approved for basal cell carcinoma, although resistance and downstream GLI activation remain challenges. Assessing ligand, receptor, and effector levels clarifies the route and extent of pathway activation. This pack brings together validated antibodies against Shh, Patched1, Indian Hedgehog / IHH, Gli1, Gli2, SUFU, KIF7, and Gli3 for studying Hedgehog signaling.