The Hippo pathway controls organ size and maintains tissue homeostasis through a central MST–LATS kinase cascade. When Hippo signaling is on, activated MST1/2 partner with SAV1 to phosphorylate and activate the LATS1/2–MOB1 complexes, which in turn phosphorylate and inactivate YAP/TAZ transcription co-activators. This process halts the expression of Hippo-responsive genes, thereby inhibiting cell proliferation and promoting apoptosis. Our studies have shown that two core adaptor proteins MOB1 and SAV1 use distinctive mechanisms to enhance Hippo signaling. MOB1 promotes MST-dependent LATS activation through dynamic scaffolding and allosteric regulation. SAV1 promotes MST activation by antagonizing the PP2A phosphatase activity. Here we describe the detailed methods for the purification and crystallization of the MST2–SAV1 and pMOB1–LATS1 complexes, for assaying the SAV1-dependent inhibition of PP2A, and for analyzing LATS1 kinase activation using in vitro reconstitution.