One major advantage of acutely dissociated inferior colliculus (IC) neurons in electrophysiological investigations is their complete isolation from the surrounding cellular network. In this way, patch-clamp recordings can be performed under controlled conditions to study membrane properties of IC neurons in more detail. The aim of the present study was to adapt a dissociation method for immature IC neurons to the highly sensitive, fragile and vulnerable mature IC neurons of mammals (mice). The modification of a pronase-based dissociation protocol with respect to concentration, incubation time and handling (trituration) of the cells yielded intact, live IC neurons with a clean cell surface so that they were well suited for further electrophysiological investigations in our study. The largely modified dissociation protocol is described in detail and critically discussed.