Aminoglycosides are broad‐spectrum antibiotics used in the management of severe infections. Aminoglycosides are associated with nephrotoxicity and ototoxicity. Although dosing strategies such as once‐daily administration and therapeutic drug monitoring have reduced the incidence of nephrotoxicity, ototoxicity remains unpredictable and may occur at therapeutic concentrations. A strong association between specific mitochondrial DNA variants in MT‐RNR1 (m.1555A > G, m.1494C > T and m.1095 T > C) and aminoglycoside‐induced hearing loss exists. These variants (frequency ~1 in 330 individuals across populations) predispose to irreversible, sensorineural hearing loss following aminoglycoside exposure, sometimes after a single dose. Avoidance of aminoglycosides is recommended at any detectable variant level. In England, laboratory‐based MT‐RNR1 testing is nationally commissioned, whereas point‐of‐care testing in time‐critical settings like neonatal sepsis is delivered in some centres. Approximately 20% of aminoglycoside use is predictable providing opportunities for pre‐emptive pharmacogenetic testing. Where MT‐RNR1 testing results are unavailable and clinical urgency is high, aminoglycoside treatment should not be delayed. Early health economic evidence suggests that point‐of‐care testing in neonates may be cost‐saving by preventing lifelong hearing loss. Regulatory and Health Technology Assessment bodies support targeted implementation of testing alongside further evidence generation. Overall, integration of MT‐RNR1 pharmacogenetic testing offers a feasible and proportionate strategy to reduce harm while preserving access to life‐saving antibiotic therapy. This guideline is grounded in the latest evidence in this field but cannot account for all individual factors relevant to patient care. Therefore, prescribers must conduct a thorough assessment of each patient's risk–benefit profile, ensuring that therapy is optimized to maximize benefits while minimizing potential harms.