Salmeterol is a man-made beta-2-adrenergic receptor agonist used to relieve bronchospasm associated with inflammatory airway disease in horses. Whilst judicious use is appropriate in horses in training, they cannot race with clinically effective concentrations of medications under the British Horseracing Authority's Rules of Racing. Salmeterol must therefore be withdrawn prior to race day and pharmacokinetic (PK) studies used to establish formal detection time advice. Salmeterol xinafoate (Serevent Evohaler® ) was administered (0.1 mg twice daily for 4.5 days) via inhalation to six horses. Urine and blood samples were taken up to 103 h postadministration. Hydrolysed samples were extracted using solid phase extraction. A sensitive Ultra high performance tandem mass spectrometry (UPLC-MS/MS) method was developed, with a Lower limit of quantification (LLOQ) for salmeterol of 10 pg/mL in both matrices. The majority of salmeterol plasma concentrations, postlast administration, were below the method LLOQ and so unusable for PK analysis. Urine PK analysis suggested a half-life consistent with duration of pharmacological effect. Average estimated urine concentration at steady-state was obtained via PK modelling and used to estimate a urine concentration of 59 ± 34 pg/mL as a marker of effective lung concentration. From this, potential detection times were calculated using a range of safety factors.