The goal of this investigation was to establish a reliable technique for the quantitation of ponazuril in limited sample volumes. Samples were extracted with chloroform and separation was achieved with a Symmetry RP18 column. Ultraviolet absorption was measured at 254 nm. A combination of 0.1% formic acid and acetonitrile (50:50) was used as the mobile phase. The calibration curve was linear from 0.1-25 μg/mL, with a lower limit of quantification of 0.1 μg/mL with a 100 μL sample. The precision and accuracy were well within the range set by the Food and Drug Administration and the recovery was over 95%. This technique was used to analyze ponazuril samples and found to be appropriate for pharmacokinetic studies.