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Summary: The inner ear of humans and large animals is embedded in a thick and dense bone that makes dissection challenging. Here, we present a protocol that enables three-dimensional (3D) characterization of intact inner ears from large-animal models. We describe steps for decalcifying bone, using solvents to remove color and lipids, and imaging tissues in 3D using confocal and light sheet microscopy. We then detail a pipeline to count hair cells in antibody-stained and 3D imaged cochleae using open-source software.For complete details on the use and execution of this protocol, please refer to (Moatti et al., 2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.