We describe a protocol for the production of mice carrying N-ethyl-N-nitrosourea (ENU) mutations and their screening for auditory and vestibular phenotypes. In comparison with the procedures describing individual phenotyping tests, this protocol integrates a set of tests for the comprehensive determination of the causes of hearing loss. It comprises a primary screen of relatively simple auditory and vestibular tests. A variety of secondary phenotyping protocols are also described for further investigating the deaf and vestibular mutants identified in the primary screen. The screen can be applied to potentially thousands of mutant mice, produced either by ENU or other mutagenesis approaches. Primary screening protocols take no longer than a few minutes, apart from ABR testing which takes upto 3.5 h per mouse. These protocols have been applied for the identification of mouse models of human deafness and are a key component for investigating the genes and genetic pathways involved in hereditary deafness.