Meldonium is a metabolic modulator, sold under the brand name Mildronate. It is used in the treatment of cerebral and cardiac ischemic disorders, owing to its ability to dilate blood vessels and increase blood flow resulting in increased oxygen flow to the cells. Within sport, evidence has indicated the capability of meldonium to increase endurance and to improve recovery time following exercise, and it is therefore prohibited in the majority of human and equine sports. Pharmacokinetics and detection of meldonium have previously been assessed in horses following a single oral administration, at various doses. The aim of this study was to assess its detection in plasma, urine and hair following a multidose administration (10 mg/kg once a day for 5 days) to two Thoroughbred horses. Target extraction and LC-MS/MS methods utilising hydrophilic interaction chromatography (HILIC) were developed for semi-quantitative analysis of meldonium in plasma and urine and for qualitative analysis of meldonium in hair. Sample analysis results will be presented herein.