Anti-diabetics such as sulfonylurea and thiazolidinedione derivatives are hypoglycemic drugs used for the treatment of diabetes. However, they can also be used as a stopper in horseracing. This paper describes a convenient method for the separation and simultaneous detection of 10 anti-diabetic drugs (namely glipizide, glibenclamide, glimepiride, gliclazide, tolazamide, tolbutamide, nateglinide, repaglinide, rosiglitazone and pioglitazone) in equine plasma and urine by LC-MS-MS. The anti-diabetics were isolated from equine plasma and urine by liquid-liquid extraction with 1,2-dichloroethane at acidic pH, and analysed by LC-MS-MS in the positive electrospray ionisation mode. Separation of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a mixture of aqueous ammonium formate (pH 3.0, 10 mM) and methanol as the mobile phase. Detection and confirmation of the 10 anti-diabetic drugs at 10 ng/mL each in equine plasma and equine urine were achieved by full-scan MS-MS. All of these drugs were detected consistently at this concentration in spiked samples of different plasma and urine (n = 15 each). No significant matrix interferences were observed at the expected retention times of the targeted ions in blank urine samples (n = 30). This method has been used successfully in the analysis of drug-administration samples as well as official racing samples. An LC-MS-MS method has been developed for the simultaneous detection of 10 anti-diabetics in equine plasma and urine. This method can be used to detect the abuse of anti-diabetic drugs in racehorses.