The 2023 Prohibited List issued by the World Anti-Doping Agency (WADA) permits athletes to inhale beta₂-agonist vilanterol at a standard dose of 25 μg daily. However, given limited data on urine pharmacokinetics, vilanterol has no urinary threshold or decision limit to discriminate therapeutic from supratherapeutic use. We investigated urine concentrations of vilanterol and its main metabolites GSK932009 and GW630200 over 0-72 hours following inhalation of therapeutic (25 μg) or supratherapeutic (100 μg) doses and repeat-dose administration for 7 days of 25 or 100 μg×day^-1 in 25 trained men and women. Vilanterol administration was followed by 1 hour of exercise. GW630200 urine concentrations were low and insufficient for threshold purposes, and while GSK932009 had higher urine concentrations, it could not discriminate between therapeutic and supratherapeutic use. Mean (range) maximum urine concentrations of parent vilanterol were 1.2(0.2-4.1) and 6.2(1.4-14.3) ng×mL^-1 for single-dose 25 and 100 μg vilanterol, respectively, and 2.0(0.3-4.8) and 22.4(6.4-42.1) ng×mL^-1 for repeat-dose 25 and 100 μg×day^-1 vilanterol. In 333 samples collected 6 hours post-administration and considering WADA TD2022DL, a 3.1 ng×mL^-1 vilanterol cut-off showed a 30% sensitivity in detecting supratherapeutic use at 100 μg versus therapeutic use at 25 μg. Considering inter- and intra-individual variability and guard bands in doping analysis, a 6 ng×mL^-1 decision limit, which could be shifted upwards in samples with specific gravity >1.018, appears sufficiently high to minimize risk of samples exceeding the decision limit after therapeutic use of vilanterol, while demonstrating the ability to detect supratherapeutic use at 100 μg.