Pergolide, an ergot-derived dopamine D2 receptor agonist, is used extensively as an orally administered treatment for pituitary pars intermedia dysfunction (PPID) in horses. One of the barriers associated with pergolide determinations in plasma for pharmacokinetic applications has been the technically demanding requirement for sensitivity. The objective of our work was to develop a simple assay for the determination of pergolide in plasma and demonstrate its potential application in the study of pergolide pharmacokinetics (PK) in horses. A UPLC–MS/MS assay was developed with a simple sample preparation involving methanol protein precipitation and injection of supernatant. The assay was applied to samples from a horse dosed with 10 mg pergolide (as the mesylate salt) by nasogastric intubation. Plasma samples were collected over a 48 h period. The assay demonstrated performance sufficient to enable application to low level PK studies. Within-batch precision and accuracy were within acceptance criteria; precision was less than 10% RSD (n = 5) and accuracy was −7.3% at 0.014 ng/mL, the lower limit of quantification was 0.006 ng/mL and the method detection limit was 0.002 ng/mL. In the treated horse, Cmax was 0.40 ng/mL and the assay easily allowed determination of plasma levels in the elimination phase to 48 h. In conclusion, this assay using UPLC–MS/MS and methanol protein precipitation easily meets the challenging demands of pergolide analyses in plasma.