There is no consensus in the literature as to the best platelet-rich plasma (PRP) acquisition method or the ideal platelet concentration to stimulate tissue repair. Besides that, most studies do not provide a clear and replicable description of the methods used, which makes standardization and result comparison difficult. Thus, this study aimed to accurately describe the method and evaluate factors influencing PRP preparation in equines. In this regard, two protocols were used, P1 and P2, based on two centrifugation methods, which differed in blood volume, speed and time, and platelet pipetting and resuspension. In conclusion, factors such as centrifugation strength and time are essential to obtain PRP with adequate platelet count. However, other factors such as tube type and plasma pipetting and resuspension methods directly interfere with the replicability of the technique, and hence influencing PRP applicability.