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Sulfadimethoxine (SDM) was extracted from skunk serum and isolated by reversed-phase high performance liquid chromatography. SDM was detected by ultra-violet absorbance at 270 nm and quantified by comparison to an external calibration standard. Recovery data were determined by analyzing SDM fortified control serum. The overall mean recovery with relative standard deviations of SDM in fortified skunk serum samples was 99±7%. The recovery for 0.38, 5.2, and 14.2 μg/mL SDM was 96.0±7.5%, 102±6.1%, and 97.3±5.1%, respectively. The method limit of detection for SDM in skunk serum ranged from 0.032 to 0.057 μg/mL SDM with a mean value of 0.040 mg/mL SDM. The method reported is much simpler and equally efficient as previous methods developed for the determination of SDM residues in serum.