Purification of hair bundles from inner-ear organs allows biochemical analysis of bundle constituents, including proteins and lipids. We describe here the "twist-off" method of bundle isolation, where dissected inner-ear organs are embedded in agarose, then subjected to a mechanical disruption that shears off bundles and leaves them in agarose blocks. With care in the dissection and in clean-up of the isolated bundles, contamination from cell bodies can be kept to a minimum. Isolated bundles can be analyzed by a variety of techniques, including immunocytochemistry, SDS-PAGE, immunoblotting, and mass spectrometry.