Introduction: Platelet-rich plasma (PRP) is an autologous blood-derived product with several therapeutic applications. Platelet growth factors have been shown to stimulate healing and reduce pain. Its use in the equine industry, particularly for orthopedic problems such as osteoarthritis or tendonitis has been increasing. However, PRP lacks reproducibility and, sometimes, quality since not only individual (breed, age, gender) and environmental (time of the day, hydration status) factors, but also laboratory methods can affect its final quality. Aims: we aimed to develop and normalize a protocol for PRP preparation, which will be repeatable for equine use, through a sterile disposable kit with a standard centrifuge, which minimizes laboratory variants. Methods and results: The final protocol resulting in a PRP with very high platelet concentration (6.4 ± 1.3 fold increase) and low white blood cell counts (3.7 ± 1.8 x106/mL) was stablished. The usual fold increase reported is 2 to 3. The final PRP had concentrations of platelet-derived growth factor with an average of 37ng/ml. Conclusion: The protocol is highly repeatable and simple to perform and the kit is suitable to use under field conditions because it does not permit contact between the sample and ambient air along the process.