Platelet-rich plasma (PRP) can be widely used in veterinary medicine in different areas. Studies using PRP frequently use different methodologies making for difficult comparison. The objective of this study was to evaluate the purity and platelet activation of a PRP protocol. A total of 18 blood samples were drawn from six dogs, collected once per week over a total of three weeks. Blood samples were centrifuged six times at 300g for 5 min. Ultra-pure PRP (OP) was obtained by adding PRP a Optiprep 1.063g/mL density barrier and centrifuged at 350g for 15 min. Mean platelet recovery from whole blood was 62.90% in PRP and 45.24% in OP. PRP and OP showed high platelet purity; blood cell contamination <0.01%. Flow cytometry for platelet activation markers was consistent with minimal platelet activation. This study describes the optimization of PRP protocol with high platelet purity, minimal platelet activation, high reliability and reproducibility.