Objective: To identify centrifugation parameters that maximize platelet recovery while preserving function in canine PRP. Methods: Whole blood from 10 healthy dogs was processed by single-spin (10 min at 800, 1,200, 1,500, or 2,000 rpm) or dual-spin (two-time immediate sequential repetition of the same centrifugation speed for 5 min) using a fixed-angle rotor (radius = 15 cm; 1,500 rpm = 364 × g equivalent). Platelet recovery and white blood cell (WBC) contamination were quantified, and platelet aggregation and dense-granule secretion (2-MeSADP, thrombin), thromboxane (Tx) A2 generation (TxB 2 enzyme-linked immunosorbent assay [ELISA]), protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) phosphorylation (immunoblot), and platelet-derived growth factor (PDGF)-BB release (ELISA) were measured. Results: Among single-spin conditions, PRP obtained at 1,500 rpm/10 min yielded the highest platelet recovery (≈25.1%) with < 1% WBC carryover. This PRP exhibited maximum 2-MeSADP- and thrombin-induced platelet aggregation and secretion, TxA2 generation, AKT/ ERK phosphorylation, and PDGF-BB release, suggesting that optimizing the centrifugal speed is crucial for achieving the optimal PRP collection with the utmost platelet function.Compared with the optimal single-spin condition, dual-spin at 1,500 rpm/5+5 min further increased platelet recovery (≈48.7%) and enhanced functional readouts. Conclusions and Relevance: A dual-spin at 1,500 rpm/5+5 min protocol produces leukocytepoor PRP with superior platelet recovery and function in dogs, supporting more effective and reproducible clinical and research applications.