The present study investigated the influence of exercise and dehydration on the urine concentrations of salbutamol after inhalation of that maximal permitted (1600 µg) on the 2015 World Anti-Doping Agency (WADA) prohibited list. Thirteen healthy males participated in the study. Urine concentrations of salbutamol were measured during three conditions: exercise (EX), exercise+dehydration (EXD), and rest (R). Exercise consisted of 75 min cycling at 60% of VO2max and a 20-km time-trial. Fluid intake was 2300, 270, and 1100 mL during EX, EXD, and R, respectively. Urine samples of salbutamol were collected 0-24 h after drug administration. Adjustment of urine concentrations of salbutamol to a specific gravity (USG) of 1.020 g/mL was compared with no adjustment. The 2015 WADA decision limit (1200 ng/mL) for salbutamol was exceeded in 23, 31, and 10% of the urine samples during EX, EXD, and R, respectively, when unadjusted for USG. When adjusted for USG, the corresponding percentages fell to 21, 15, and 8%. During EXD, mean urine concentrations of salbutamol exceeded (1325±599 ng/mL) the decision limit 4 h after administration when unadjusted for USG. Serum salbutamol Cmax was lower (P<0.01) for R(3.0±0.7 ng/mL) than EX(3.8±0.8 ng/mL) and EXD(3.6±0.8 ng/mL). AUC was lower for R (14.1±2.8 ng/mL·∙h) than EX (16.9±2.9 ng/mL·∙h)(P<0.01) and EXD (16.1±3.2 ng/mL·∙h)(P<0.05). In conclusion, exercise and dehydration affect urine concentrations of salbutamol and increase the risk of Adverse Analytical Findings in samples collected after inhalation of that maximal permitted (1600 µg) for salbutamol. This should be taken into account when evaluating doping cases of salbutamol. Copyright © 2015 John Wiley & Sons, Ltd.