Pure-tone thresholds have long served as a gold standard for evaluating hearing sensitivity and documenting hearing changes related to medical treatments, toxic or otherwise hazardous exposures, ear disease, genetic disorders involving the ear, and deficits that develop during aging. Although the use of pure-tone audiometry is basic and standard, interpretation of thresholds obtained at multiple frequencies in both ears over multiple visits can be complex. Significant additional complexity is introduced when audiometric tests are performed within ototoxicity monitoring programs to determine if hearing loss occurs as an adverse reaction to an investigational medication and during the design and conduct of clinical trials for new otoprotective agents for noise and drug-induced hearing loss. Clinical trials using gene therapy or stem cell therapy approaches are emerging as well with audiometric outcome selection further complicated by safety issues associated with biological therapies. This review addresses factors that must be considered, including test-retest variability, significant threshold change definitions, use of ototoxicity grading scales, interpretation of early warning signals, measurement of notching in noise-induced hearing loss, and application of age-based normative data to interpretation of pure-tone thresholds. Specific guidance for clinical trial protocols that will assure rigorous methodological approaches and interpretable audiometric data are provided.