Background: Developing manual platelet-rich plasma (PRP) methods in donkeys improves cost-efficient field management in a species often treated under resource limitations. Objectives: To compare asinine PRP manually produced by single and double centrifugation. Study design: Ex vivo experimental study. Methods: Whole blood (WB) from 6 healthy donkeys was collected into sodium citrate vacutainer tubes for single centrifugation processing and an acid-citrate-phosphate-dextrose-adenine blood bag for double centrifugtation processing to produce, respectively, PRP1 and PRP2. Concentrations of platelets ([PLT]), leukocyte ([WBC]), and erythrocytes ([RBC]), and activities of platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) were assessed in WB, PRP1, PRP2. Results were compared by Friedman’s and Dunn’s multiple comparison test. Spearman’s rank was used to assess the correlation between [PLT] in WB, PRP1, and PRP2. Results: Both centrifugation protocols concentrated platelets 1.8–5.2-fold, reduced WBC 1.1–50.4-fold, and decreased RBC at least 829-fold compared to WB. PRP2 yielded a higher [PLT] and platelet enrichment factor than PRP1. However, PRP 1 possessed lower [WBC] and greater WBC reduction factor than PRP2. PDGF-BB and TGF-β1 activities were higher in PRP2 than PRP1 and WB. Growth factor activities were not significantly different between PRP1 and WB. There was weak and moderate correlation of baseline [PLT] to that of PRP2 (r = 0.4) and PRP1 (r = 0.62), respectively; neither was statistically significant ( p > 0.05). PRP2 yielded higher platelet enrichment and growth factor activities despite greater WBC and RBC contamination than PRP1. Main limitations: Non-randomized collection methods. Conclusions: Manual, noncommercial methods of equine PRP preparation can be used to produce PRP in donkeys. Double centrifugation yielded a more concentrated platelet product with higher leukocyte contamination than single centrifugation. The clinical utility of leukocyte contaminated PRP and its optimal platelet concentration, in vivo efficacy, and safety warrent further investigation.