Gentamicin is an aminoglycoside antibiotic effective against aerobic gram-negative bacteria and is also used in veterinary medicine, particularly in the swine and bovine industries. However, no gentamicin product is currently approved for treating equine diseases in Korea. The present study aims to examine the time-dependent residue of gentamicin in horses after intravenous injection (IV) via jugular vein. The test product was injected at 6.6 mg/kg BW via jugular vein in nine horses. Blood was collected from the horse's jugular vein at 15 minutes, 30 minutes, 1, 4, 8, 12, 24 and 48 hours after injection. To purify the gentamicin in serum, 100μL of 20 mM HFBA in DW, 100 μL of 30% trichloroacetic acid and 300 μL of 20 mM heptafluorobutyric acid (HFBA) in acetonitrile (ACN) were added to 500 μL of serum and supernatant was applied to LC-MS/MS after centrifugation. LC-MS/MS-8050 analyzed the level of gentamicin in serum with Electrospray ionization (ESI) and multiple reaction monitoring (MRM) positive mode. Gentamicin C1 was 478 m/z and product ions were 322, 157 m/z. Precursor ion of Gentamicin C1a was 450 m/z and product ions were 322, 160 m/z. Precursor ion of Gentamicin C2 and C2a was 464 m/z and product ions were 322, 160 m/z. The LC column was a C18 and mobile phase composed of 20 mM HFBA in 5% ACN and 20 mM HFBA in 50% ACN. The amount of gentamicin was calculated by adding four components of gentamicin (C1, C1a, C2 and C2a). The pharmacokinetic parameters of gentamicin were calculated by the WinNonlin program. The Cmax of gentamicin in horse serum was 93 ± 17 μg/kg and the Tmax was 0.25 ± 0 hours. The T1/2 was 6.41 ± 2.32 hours and the CLt was 0.05 ± 0.01L/hr/kg. The Vd was shown as 0.44 ± 0.13 L/kg and the MRT was 1.98 ± 0.55 hours. In conclusion, our data provides useful pharmacokinetic parameters for gentamicin in horses following IV injection.